Proteoglycan mimetics for enhanced wound healing, angiogenesis, and vascular repair

ABSTRACT

The present disclosure provides pro-angiogenic proteoglycan mimetics that can provide a provisional, pro-angiogenic scaffold to support tissue regeneration while limiting systemic exposure to VEGF activity. These mimetics can protect a collagen matrix from rapid degradation, and in conjunction with EPCs promote angiogenesis in order to accelerate ischemic wound healing. For example, the provided compounds can be delivered from the end of a catheter following balloon angioplasty to coat the collagen exposed areas, prevent platelet binding and thrombosis, support capture of EPCs from blood to facilitate reendothelialization, and reduce late-lumen loss (neointimal hyperplasia).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/062,300, filed Oct. 2, 2020, which is a continuation of InternationalPatent Application No. PCT/US2019/026141, filed Apr. 5, 2019, whichclaims priority to U.S. Provisional Patent Application No. 62/653,329,filed Apr. 5, 2018, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. DK101001awarded by the National Institutes of Health (NIH). The Government hascertain rights in the invention.

SEQUENCE LISTING

A Sequence Listing, electronically filed herewith via Patent Center,conforming to the rules of WIPO Standard ST.26, is hereby incorporatedby reference in its entirety. The Sequence Listing .xml file, identifiedas 070772-1373612-225520US, is 76,430 bytes in size and was created onJul. 17, 2023.

BACKGROUND

According to the Centers for Disease Control and Prevention (CDC) 2014National Diabetes Statistics Report, between 15-25% of the approximately29 million diabetic Americans develop serious foot ulcers. Approximately65% of these ulcers have ischemic pathology and become chronic woundsdue to impaired healing (Armstrong et al. (2011) J. Diabetes Sci.Technol. 5:1591). To improve healing of these chronic ischemic ulcers,researchers have sought to increase angiogenesis at the wound site usingangiogenic growth factors. One key angiogenic mediator is the vascularendothelial growth factor (VEGF), which functions by improving theformation of granulation tissue, a loose fibrovascular tissue, duringthe early stages of the healing response. For example, in ischemicwounds, exogenous VEGF is known to increase granulation tissue formationup to 150% (Corral et al. (1999) Arch. Surg. 134:200). However, theclinical success of such growth factor therapies for chronic wounds hasbeen limited largely due to the overexpression of matrixmetalloproteases (MMPs) that can degrade or inactivate the growthfactors in the wound environment (Fang and Galiano (2008) Biol. TargetsTher. 2:1). Excess MMPs can also promote the degradation of newly formedgranulation tissue, thus counteracting healing. Furthermore,non-targeted angiogenic growth factor therapies are not restricted tothe wound site, and can potentially diffuse into systemic circulationand cause malignancies.

Coronary atherosclerosis is currently the leading cause of death insociety. Moreover, atherosclerotic conditions in an individual can begreatly exacerbated by the presence of risk factors such as metabolicsyndrome (“pre-diabetes”), which itself often progresses to full-blowntype-2 diabetes. Percutaneous coronary intervention (PCI) procedures arecommonly performed in coronary arteries to open atherosclerotic oroccluded vessels which would otherwise lead to ischemia if leftuntreated. PCI treatment involves threading a balloon catheter to theoccluded vessel, whereupon the balloon is deployed to widen the vessel.A typical consequence of this procedure is damage to the vessel wall atthe site of balloon deployment. The layer of endothelial cells (ECs)covering the internal lumen of the vessel can be stripped, and theunderlying collagenous connective tissue can become exposed, initiatingcoagulation and inflammatory cascades of the body of the patient undertreatment. These cascade responses within the patient's body in turntrigger thrombus formation, inflammation, smooth muscle cell (SMC)proliferation and migration, and extracellular matrix deposition.Together these effects can negatively impact both the safety andefficacy of the procedure. Left untreated, the lesions ofatherosclerosis result in coronary arterial stenosis (narrowing) orblockage and consequent disruption of blood flow, culminating inmyocardial infarction (heart attack) and death.

When conventional balloon angioplasty alone is used to open blockedvessels, in greater than 50% of cases the reopened artery closed againdue to restenosis after the angioplasty. As a result, the patient withrestenosis, or overgrowth of SMCs, often will require a secondangioplasty or emergency bypass graft surgery. In part to address thesecomplications, metallic coronary stents (wire meshes) were developed andintroduced in 1993. While these stents have been effective in preventingabrupt artery closure due to vasoconstriction, serious injury to theblood vessels continue to occur, causing neointimal proliferation andleading to in-stent restenosis and the need for revascularization inapproximately 20% of patients. This overgrowth of SMCs after surgery issimilar to scar tissue formation in response to injury.

The problem of in-stent restenosis has been somewhat reduced by theintroduction of drug-eluting stents (DESs) in 2003. For example, inearly evaluations, sirolimus-eluting stents (CYPHER™ stents from Cordisin 2003) and PTX-eluting stents (TAXUS™ stents from Boston Scientific in2004) have shown effective inhibition of neointimal hyperplasia andprevention of restenosis in single de novo coronary lesions, withrevascularization only required in approximately 10% of patients. In2008, the Food and Drug Administration (FDA) approved two more DESdevices, Medtronic's ENDEAVOR® stent delivering zotarolimus, and AbbottLaboratories' XIENCE™ V stent delivering everolimus. However, recentevidence shows that ten years post-angioplasty, the performance of DESdevices may be statistically no better than that of bare metal stents.

In addition to suggesting that DES devices do not improve outcomesrelative to the use of bare metal stents at later time points, recentdata indicates that that the DES devices may be susceptible to bothearly and late in-stent thrombosis. Early stage thrombosis is generallydue to initial denudation of ECs and exposure of the underlyingcollagen. Late stent thrombosis is characterized by the persistence offibrin and incomplete endothelialization at time points beyond 30 daysfrom the time of stenting. These partially endothelialized, fibrin-richsites provide a stimulus for surface-induced thrombosis, with thepresence of activated platelets possibly helping to exacerbateinflammation. Importantly, even if the incidence of thrombosis is low,such conditions are still of great concern as 90% of all thromboticevents lead to death. Additionally, late stent thrombosis has beenassociated with an increased frequency of other chronic maladies,including renal insufficiency, decreased left ventricular function, anddiabetes. Accordingly, patients having stent thrombosis are typicallyprescribed antiplatelet medications, such as aspirin and PLAVIX® orTICLID®, indefinitely.

The near epidemic of pre-diabetes and progression of patients to type 2diabetes threatens to compound coronary disease morbidity and mortality,increasing the need for coronary stenting, and increasing the incidenceof stent thrombosis. In addition, diabetes can lead to serious woundsites such as those of foot ulcers as described above. In view of this,there is a need in the art for materials and methods that can provideenhanced wound healing, angiogenesis, and vascular repair in diversemedical applications. The present disclosure satisfies this need andprovides other advantages as well.

BRIEF SUMMARY

One provided compound comprises: one or more P1 subunits, wherein P1 isa synthetic peptide comprising an amino acid sequence that comprises acollagen-binding domain; one or more P2 subunits, wherein P2 is asynthetic peptide comprising an amino acid sequence that comprises anintegrin-binding domain; and a glycan, wherein each P1 subunit and eachP2 subunit is linked to the glycan. In some embodiments, P1 is asynthetic peptide comprising an amino acid sequence having at least 80%sequence identity with the amino acid sequence RRANAALKAGELYKSILY(SEQ IDNO: 1). In some embodiments, P1 is a synthetic peptide of up to about 40amino acids comprising the amino acid sequence RRANAALKAGELYKSILY(SEQ IDNO: 1). In some embodiments, P2 is a synthetic peptide comprising anamino acid sequence that comprises an αvβ3-binding domain. In someembodiments, P2 is LXW7. In some embodiments, P2 is peg2V. In someembodiments, the glycan is a glycosaminoglycan or polysaccharide. Insome embodiments, the glycan is selected from the group consisting ofalginate, agarose, dextran, chondroitin, dermatan, dermatan sulfate,heparan, heparin, keratin, and hyaluronan. In some embodiments, theglycan is selected from the group consisting of dermatan sulfate,dextran, and heparin.

Also provided is a compound comprising: one or more P1 subunits, whereinP1 is a synthetic peptide comprising an amino acid sequence thatcomprises a collagen-binding domain; and one or more P2 subunits,wherein P2 is a synthetic peptide comprising an amino acid sequence thatcomprises an integrin-binding domain. In some embodiments, P1 is asynthetic peptide comprising an amino acid sequence having at least 80%sequence identity with the amino acid sequence RRANAALKAGELYKSILY(SEQ IDNO: 1). In some embodiments, P1 is a synthetic peptide of up to about 40amino acids comprising the amino acid sequence RRANAALKAGELYKSILY(SEQ IDNO: 1). In some embodiments, P2 is a synthetic peptide comprising anamino acid sequence that comprises an αvβ3-binding domain. In someembodiments, P2 is LXW7.

Also provided is a composition comprising any of the disclosed compoundsdescribed above and one or more pharmaceutically acceptable excipients,diluents, or a combination thereof.

Also provided is a method for improving endothelialization andvascularization of endothelial cells and/or endothelial progenitor cellsin a subject, the method comprising administering one or morecompositions, each comprising one or more of any of the disclosedcompounds described above to the subject. In some embodiments, at leastone of the one or more compositions further comprises one or morepharmaceutically acceptable excipients, diluents, or a combinationthereof. In some embodiments, the subject is a patient suffering from adisease associated with endothelial dysfunction. In certain aspects, thedisease is an ulcer. In some embodiments, the subject is in need ofcoronary stenting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the synthetic scheme of SILY-DBCO.

FIG. 2 (SEQ ID NO: 7) illustrates the synthetic scheme of LXW7-2N3.

FIG. 3 illustrate the synthetic scheme of LXW7-(SILY)₂.

FIG. 4 illustrates the synthetic scheme of LXW7-DBCO.

FIG. 5 (SEQ ID NO: 8) illustrate the synthetic scheme of SILY-2N3.

FIG. 6 illustrates the synthetic scheme of SILY-(LXW7)₂ conjugate.

FIG. 7 presents images and a graph of human endothelial colony formingcells (HECFCs) binding on collagen surface (5 min).

FIG. 8 presents graphs of the effect of LXW7-SILY on EC apoptosisinduced by hypoxia.

FIG. 9 presents images of EC attachment on SIS scaffold with LXW7-SILY.

FIG. 10 presents images of EC proliferation and organization on SISscaffolds modified with LXW7-SILY.

FIG. 11 presents a graph of platelet binding with LXW7-DS-SILY.

FIG. 12 presents a graph of platelet binding with LXW7-(SILY)₂.

FIG. 13 presents graphs of circular dichroism spectra measured todetermine any peptide conformational changes.

FIGS. 14A and 14B presents graphs of 13C and 1H NMR spectra.

FIG. 15 illustrates the reaction scheme of O-acylisourea rearrangementto stable N-acylurea after carboxylic acid activation.

FIG. 16 presents absorbance graphs of binding.

FIG. 17 presents graphs of microvascular endothelial cell adhesion tosurface-bound molecules

FIG. 18 presents graphs of endpoint MTS metabolic assay results.

FIG. 19 presents graphs of endothelial proliferation over time forDS-SILY₄ and (peg2V)₃-DS-SILY₄.

FIG. 20 presents graphs of endothelial proliferation over time forLXW7-DS-SILY₄ variants.

FIG. 21 presents graphs of endpoint results of endothelial proliferationusing LXW7-DS-SILY₄ variants.

FIG. 22 presents graphs and images of migration responses tosurface-bound molecules.

FIG. 23 presents graphs of matrigel tubulogenesis assay results using 10μM treatments and untreated controls.

FIG. 24 presents graphs and images of matrigel tubulogenesis assayresults using 20 μM treatments.

FIG. 25 presents a graph of chick chorioallantoic membranevascularization in response to collagen gels with 100 μM peptides.

FIG. 26 presents graphs of chick chorioallantoic membranevascularization in response to collagen gels with 10 μM or 20 μMpeptides.

FIG. 27 presents images of representative vascularized gels.

FIG. 28 presents images of vasculature invading implanted collagen gelsvisualized with high molecular weight fluorescent dextran.

FIG. 29 presents graphs of the effects of peptides on endothelialpermeability.

FIG. 30 . EC-SMC co-culture system for evaluation of therapeutics. SMCswere tracked via CellTracker™ Green CMFDA and ECs were tracked byspecifically labeling them with DiI-Ac-LDL. In image B SMCs are labeledwith α-SMA (Green) and ECs are labeled with vWF (red).

FIG. 31 . Following endothelial denudation by balloon angioplasty inOssabaw pigs, vessels were treated with saline (left) or DS-SILY(right). The saline treated vessel had high amounts of platelet binding(arrows in top left) while the DS-SILY treated vessel almost completelyinhibited platelet binding (top right). Platelets that were present inthe DS-SILY treated vessel remained rounded (bottom right) indicatingthey were not activated. Conversely, the platelets on the saline treatedvessel had many protrusions (arrows in bottom left), indicatingactivation.

FIG. 32 . Representative histology sections of (A, B) arteries withstents and (C, D) arteries without stents treated with (A, C) saline or(B, D) 10 μM DS-SILY20. Arrowheads indicate location of some stentstruts; internal elastic lamina (dotted line) and luminal border (solidline) are identified, indicating the boundaries of the neointima formedfollowing injury. (E) Neointimal hyperplasia was quantified by measuringthe distance from a stent strut or the elastic lamina to the vessellumen in arteries with or without stents, respectively. Analysis withstents: sham (n=4), DS-SILY20 (n=3); without stents: sham (n=8),DS-SILY20 (n=5). Scale bar=1 mm.* represents significance fromsham-treated vessels.

FIG. 33 . Attachment of cells and platelets to LXW7 and GRGD (SEQ ID NO:2) treated surfaces. (A) Images of attached HCAECs (left panels), THP-1monocytes (middle panels) and platelets (right panels) on surfacestreated by D-Biotin (a-c) (control), LXW7 (d-f) or GRGD (SEQ ID NO: 2)(g-i). Scale bars in a, b, d, e, g and h are 50 μm. Scale bars in c, fand i are 20 μm. (B) The number of cells or platelets attached ondifferent treated surfaces were quantified and statistical analyses wereperformed. Data were expressed as mean±standard deviation: **p<0.01(n=4).

FIG. 34 . Effects of LXW7 on EC biological functions. (A) Proliferationof ECs on LXW7-treated surfaces and D-biotin treated surface (control)assessed by MTS assay. Data were expressed as mean±standard deviation:**p<0.01, ***p<0.001 (n=4). (B) Western-blot analysis of the effect ofLXW7 on phosphorylation of VEGFR2 (Tyr1175) and phosphorylation ofERK1/2 (left panels) and quantified by densitometry (right panels). Datawere expressed as mean±standard deviation: *p<0.05, **p<0.01 (n=4).

FIG. 35 . Patency study of LXW7 conjugation on the luminal surface ofsmall diameter vascular grafts increased endothelialization of the in arat carotid artery bypass model. Representative images of (A) thrombosed(control grafts without LXW7 modification) and (B) patent grafts(LXW7-modified) at 6 weeks. Temporal patency analysis (C) showed thatLXW-modified grafts maintained significant higher patency rate than theunmodified control grafts.

DETAILED DESCRIPTION I. General

The present disclosure is based, in part, on the discovery thatadministration of collagen-binding, pro-angiogenic proteoglycanmimetics, such as LXW7-DS-SILY, and/or a peg2V-DS-SILY variant, canprovide a provisional, pro-angiogenic scaffold to support tissueregeneration while limiting systemic exposure to VEGF activity. Thesemimetics can protect a collagen matrix from rapid degradation, and inconjunction with EPCs promote angiogenesis in order to accelerateischemic wound healing. For example, the addition of LXW7 to the DS-SILYmolecule, (LXW7-DS-SILY), will result in a molecule that can bedelivered from the end of a catheter following balloon angioplasty tocoat the collagen exposed areas, prevent platelet binding andthrombosis, support capture of EPCs from blood to facilitatereendothelialization, and reduce late-lumen loss (neointimalhyperplasia). This disclosure can therefore provide a therapeutic thatcan selectively affect damaged areas without long-term complications ofpermanent stents including late stent thrombosis and a foreign bodyresponse that promotes scarring. This new therapeutic can alleviate theneed for long-term daily administration of anti-clotting agents. The newtherapeutic can thus substantially lower the complications associatedwith current drug-eluting stents.

The provided approach in preventing restenosis and late thrombosis isdifferent from the existing method of delivering non-specific drugs,such as sirolimus and paclitaxel. The disclosed approach is innovativein allowing suppression of platelet binding and inflammation whilepromoting reendothelialization, and eliminating the need forimplantation of a stent. In addition, the delivery is consistent withcurrent clinical practice and is agnostic to type of balloon that isused. Advantageously, the procedure causes denudation of the bloodvessel and exposes collagen, which in turn promotes platelet binding andactivation. Platelet activation can lead to thrombosis and activation ofthe inflammatory system. Temporary antithrombotic coating withLXW7-DS-SILY limits both early and late stage thrombosis andinflammation, and thus promotes proper vessel recovery. In addition, theLXW7 peptide can capture circulating EPCs through specific interactionwith the α_(v)β₃ integrin and promote reendothelialization of thedamaged vessel. LXW7 will not capture platelets, monocytes, or otherblood cells. The provided approach is also surprisingly effective ineliminating multiple limitations of current DES technology. The newapproach will block early term thrombosis, inhibit inflammation, limitSMC proliferation, and promote reendothelialization to promote vesselhealing and prevent late term thrombosis.

II. Definitions

The term “scaffold” refers to a matrix that provides a three-dimensionalstructure suitable for cell culture, tissue engineering, or tissueregeneration. The structure of a scaffold can have, for example, theform of a stent, a shunt, a patch, a graft, or an implant. Scaffolds canbe modified to promote cell recruitment, adhesion, or proliferation.Exemplary modifications include, but are not limited to, incorporationof one or more cell adhesion promoters, surface coatings, or functionalgroups.

The term “amino acid” refers to naturally-occurring α-amino acids andtheir stereoisomers, as well as unnatural amino acids and theirstereoisomers. “Stereoisomers” of amino acids refers to mirror imageisomers of the amino acids, such as L-amino acids or D-amino acids. Forexample, a stereoisomer of a naturally-occurring amino acid refers tothe mirror image isomer of the naturally-occurring amino acid, i.e., theD-amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. For example, an L-aminoacid may be represented herein by its commonly known three letter symbol(e.g., Arg for L-arginine) or by an upper-case one-letter amino acidsymbol (e.g., R for L-arginine). A D-amino acid may be representedherein by its commonly known three letter symbol with “D” as prefix(e.g., DArg, D-Arg or DArg for D-arginine) or by a lower-case one-letteramino acid symbol (e.g., r for D-arginine).

Amino acids can be characterized by at least one of several properties.For example, amino acids can be basic, acidic,-polar or hydrophobic.Basic amino acids are those having a basic or positively charged sidechain at pH values below the pKa, and include, but are not limited to,Lys, Arg, HoArg, Agp, Agb, Dab, Dap and Orn and stereoisomers thereof.Acidic amino acids are those having an acidic or negatively charged sidechain at physiological pH, and include, but are not limited to, Asp,Glu, Aad, Bec and stereoisomers thereof. Basic amino acids can generallybe referred by the symbol “X⁺” and acidic amino acids by “X⁻”. Polaramino acids generally refer to those having a polar and uncharged sidechain and include, but are not limited to, Asn, Ser, Thr, Gln, andstereoisomers thereof. Similarly, hydrophobic amino acids generallyrefer to those having a hydrophobic side chain and include, but are notlimited to, Val, Leu, Ile, Met, and Phe. One of skill in the art willappreciate that other basic and acidic amino acids are known in the art.

With respect to amino acid sequences, one of skill in the art willrecognize that individual substitutions, additions, or deletions to apeptide, polypeptide, or protein sequence which alters, adds, or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. The chemically similar amino acid includes, withoutlimitation, a naturally-occurring amino acid such as an L-amino acid, astereoisomer of a naturally occurring amino acid such as a D-amino acid,and an unnatural amino acid such as an amino acid analog, amino acidmimetic, synthetic amino acid, N-substituted glycine, and N-methyl aminoacid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, substitutions may be madewherein an aliphatic amino acid (e.g., G, A, I, L, or V) is substitutedwith another member of the group. Similarly, an aliphaticpolar-uncharged group such as C, S, T, M, N, or Q, may be substitutedwith another member of the group; and basic residues, e.g., K, R, or H,may be substituted for one another. In some embodiments, an amino acidwith an acidic side chain, e.g., E or D, may be substituted with itsuncharged counterpart, e.g., Q or N, respectively; or vice versa. Eachof the following eight groups contains other exemplary amino acids thatare conservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M)    -   (see, e.g., Creighton, Proteins, 1984)

The term “peptide” refers to a compound made up of a single chain of Dor L amino acids or a mixture of D and L amino acids joined by peptidebonds. Generally, peptides are about 2 to about 50 amino acids inlength.

The term “biopolymer” refers to either a naturally occurring polymer, ora synthetic polymer that is compatible with a biological system or thatmimics naturally occurring polymers. For example, and not by way oflimitation, biopolymers of the present disclosure includeoligosaccharides, proteins, polyketides, peptoids, hydrogels,poly(glycols) such as poly(ethylene glycol), and polylactates.

The term “ligand” refers to a molecule that selectively binds,covalently or noncovalently, to another specific molecule or to aspecific part of a molecule.

The term “bind” includes any physical or chemical attachment or closeassociation, which may be permanent or temporary.

The term “noncovalent interactions” refers to the interaction of twospecies in close proximity that does not form a covalent bond. Types ofnoncovalent interactions include, for example, hydrogen bonding, van derWaals interaction, coordination, pi-pi interaction, hydrophobicinteractions and hydrophilic interactions.

The term “covalent interaction” refers to the interaction of two speciesin close proximity that form a covalent bond.

The term “αvβ3 integrin” refers to a receptor of vitronectin. αvβ3integrin serves as a receptor for a variety of extracellular matrixproteins displaying the arginine-glycine-aspartic acid (RGD) tripeptidesequence. These proteins include vitronectin, fibronectin, fibrinogen,laminin, collagen, and Von Willibrand's factor.

The term “subject” refers to animals such as mammals, including, but notlimited to, primates (e.g., humans), cows, sheep, goats, horses, dogs,cats, rabbits, rats, mice and the like. In certain embodiments, thesubject is a human.

The term “alkyl” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated. For example, C₁-C₆alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.Other alkyl groups include, but are not limited to heptyl, octyl, nonyl,decyl, etc. Alkyl can include any number of carbons, such as 1-2, 1-3,1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6,4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can bedivalent, such as when the alkyl group links two moieties together.

The term “alkoxy” refers to an alkyl group having an oxygen atom thatconnects the alkyl group to the point of attachment. Alkoxy groupsinclude, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy,2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Thealkoxy groups can be further substituted with a variety of substituentsdescribed within. For example, the alkoxy groups can be substituted withhalogens to form a “halo-alkoxy” group.

As used herein, the term “heteroalkyl” refers to an alkyl group havingfrom 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms canalso be useful, including, but not limited to, B, Al, Si and P. Theheteroatoms can also be oxidized, such as, but not limited to, —S(O)—and —S(O)₂—. For example, heteroalkyl can include ethers, thioethers andalkyl-amines.

As used herein, the term “haloalkyl” refers to alkyl as defined abovewhere some or all of the hydrogen atoms are substituted with halogenatoms. Halogen (halo) preferably represents chloro or fluoro, but mayalso be bromo or iodo. For example, haloalkyl includes trifluoromethyl,flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro”defines a compound or radical which has at least two available hydrogenssubstituted with fluorine. For example, perfluorophenyl refers to1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to1,1,1-trifluoromethyl, and perfluoromethoxy refers to1,1,1-trifluoromethoxy.

As used herein, the term “halo-alkoxy” refers to an alkoxy group havingat least one halogen. Halo-alkoxy is as defined for alkoxy where some orall of the hydrogen atoms are substituted with halogen atoms. The alkoxygroups can be substituted with 1, 2, 3, or more halogens. When all thehydrogens are replaced with a halogen, for example by fluorine, thecompounds are per-substituted, for example, perfluorinated. Halo-alkoxyincludes, but is not limited to, trifluoromethoxy,2,2,2,-trifluoroethoxy, perfluoroethoxy, etc.

As used herein, the term “halogen” refers to fluorine, chlorine, bromineand iodine.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated Monocyclic rings include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic andpolycyclic rings include, for example, norbornane, decahydronaphthaleneand adamantane. For example, C₃₋₈ cycloalkyl includes cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

As used herein, the term “alkyl-cycloalkyl” refers to a radical havingan alkyl component and a cycloalkyl component, where the alkyl componentlinks the cycloalkyl component to the point of attachment. The alkylcomponent is as defined above, except that the alkyl component is atleast divalent in order to link to the cycloalkyl component and to thepoint of attachment. In some instances, the alkyl component can beabsent. The cycloalkyl component is as defined within. Examples ofalkyl-cycloalkyl include methylene-cyclohexane, among others.

As used herein, the term “heterocycloalkyl” refers to a ring systemhaving from 3 ring members to about 20 ring members and from 1 to about5 heteroatoms such as N, O and S. Additional heteroatoms can also beuseful, including, but not limited to, B, Al, Si and P. The heteroatomscan also be oxidized, such as, but not limited to, —S(O)— and —S(O)₂—.For example, heterocycle includes, but is not limited to,tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, piperidinyl, indolinyl, quinuclidinyl and1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

As used herein, the term “alkyl-heterocycloalkyl” refers to a radicalhaving an alkyl component and a heterocycloalkyl component, where thealkyl component links the heterocycloalkyl component to the point ofattachment. The alkyl component is as defined above, except that thealkyl component is at least divalent in order to link to theheterocycloalkyl component and to the point of attachment. In someinstances, the alkyl component can be absent. The heterocycloalkylcomponent is as defined above. Examples of alkyl-heterocycloalkylinclude methylene-piperidinyl, among others.

As used herein, the term “aryl” refers to a monocyclic or fusedbicyclic, tricyclic or greater, aromatic ring assembly containing 6 to16 ring carbon atoms. For example, aryl may be phenyl, benzyl ornaphthyl, preferably phenyl. “Arylene” means a divalent radical derivedfrom an aryl group. Aryl groups can be mono-, di- or tri-substituted byone, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy,halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-C₃-alkylene; all of which are optionally further substituted, forinstance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.Oxy-C₂-C₃-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred as aryl is naphthyl, phenyl, or phenyl mono- or di-substitutedby alkoxy, phenyl, halogen, alkyl, or trifluoromethyl; especially phenylor phenyl mono- or di-substituted by alkoxy, halogen, ortrifluoromethyl; and in particular phenyl.

Examples of substituted phenyl groups as R are, e.g. 4-chlorophen-1-yl,3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methyl phen-1-yl,4-aminomethylphen-1-yl, 4-methoxy ethyl aminomethylphen-1-yl,4-hydroxyethylaminomethylphen-1-yl,4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl,4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl,2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phen-yl,4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,4-(morpholin-1-ylmethyl)-phen-1-yl, 4-(2-methoxyethylaminomethyl)-phen-1-yl and 4-(pyrrolidin-1-ylmethyl)-phen-1-yl,4-(thiophenyl)-phen-1-yl, 4-(3-thiophenyl)-phen-1-yl,4-(4-methylpiperazin-1-yl)-phen-1-yl, and 4-(piperidinyl)-phenyl and4-(pyridinyl)-phenyl optionally substituted in the heterocyclic ring.

As used herein, the term “alkyl-aryl” refers to a radical having analkyl component and an aryl component, where the alkyl component linksthe aryl component to the point of attachment. The alkyl component is asdefined above, except that the alkyl component is at least divalent inorder to link to the aryl component and to the point of attachment. Insome instances, the alkyl component can be absent. The aryl component isas defined above. Examples of alkyl-aryl groups include, but are notlimited to, benzyl.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O orS. For example, heteroaryl includes pyridyl, indolyl, indazolyl,quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl,furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any otherradicals substituted, especially mono- or di-substituted, by e.g. alkyl,nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinylrepresents preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl representspreferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranylrepresents preferably 3-benzopyranyl or 3-benzothiopyranyl,respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, andmost preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl,thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl,benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted,especially mono- or di-substituted.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

As used herein, the term “alkyl-heteroaryl” refers to a radical havingan alkyl component and a heteroaryl component, where the alkyl componentlinks the heteroaryl component to the point of attachment. The alkylcomponent is as defined above, except that the alkyl component is atleast divalent in order to link to the heteroaryl component and to thepoint of attachment. In some instances, the alkyl component can beabsent. The heteroaryl component is as defined within. Examples ofalkyl-heteroaryl include methylene-pyridyl, among others.

III. Compounds

The compounds provided herein each include one or more syntheticcollagen-binding peptides P1, and one or more synthetic integrin-bindingpeptides P2. In some embodiments, the synthetic peptide P1 includes anamino acid sequence having at least 80% sequence identity with the aminoacid sequence RRANAALKAGELYKSILY(SEQ ID NO: 1). In certain aspects, thesynthetic peptide P1 includes the amino acid sequenceRRANAALKAGELYKSILY(SEQ ID NO: 1). Variability can be present in the P1sequence. For example, the synthetic peptide P1 can be modified by theinclusion of one or more conservative amino acid substitutions. As iswell known to those skilled in the art, altering any non-critical aminoacid of a peptide by conservative substitution should not significantlyalter the activity of that peptide because the side-chain of thereplacement amino acid should be able to form similar bonds and contactsto the side chain of the amino acid which has been replaced.Accordingly, the P1 sequence can be modified such that a sequence havingat least about 80% sequence identity, or at least about 83% sequenceidentity, or at least about 85% sequence identity, or at least about 90%sequence identity, or at least about 95% sequence identity, or at leastabout 98% sequence identity thereto is incorporated in the scaffoldcompound, provided the sequence is capable of binding to collagen.

The amino acid length of synthetic peptide P1 can be, for example,between 20 and 60, e.g., between 20 and 44, between 24 and 48, between28 and 52, between 32 and 56, or between 36 and 60. In terms of upperlimits, the amino acid length of P1 can be less than 60, e.g., less than56, less than 52, less than 48, less than 44, less than 40, less than36, less than 32, less than 32, less than 28, or less than 24. In termsof lower limits, the amino acid length of P1 can be up to 20, e.g., upto 24, up to 28, up to 32, up to 32, up to 36, up to 40, up to 44, up to48, up to 52, or up to 56. Longer lengths, e.g., greater than 60, andshorter lengths, e.g., less than 20, are also contemplated.

The synthetic peptide P2 can be a peptide ligand including anintegrin-binding domain. Peptide ligands suitable for use with thepresent disclosure can be selected to increase the attachment ofendothelial cells and/or endothelial progenitor cells to the scaffold.For example, the peptide ligand can be a compound of Formula I:

-   -   X₂, X₆, and X₇ can each independently be an amino acid, wherein        at least one of X₂, X₆, and X₇ is a D-amino acid. R¹ of formula        I can be H, C₁₋₆ alkyl, —C(O)R^(1a), or L-A. R^(1a) can be C₁₋₆        alkyl, C₁₋₆ heteroalkyl, C₁₋₆ alkyl-NH₂, C₁₋₆        alkyl-C(O)N(H)—C₁₋₆ heteroalkyl, cycloalkyl, C₁₋₆        alkyl-cycloalkyl, heterocycloalkyl, C₁₋₆ alkyl-heterocycloalkyl,        aryl, C₁₋₆ alkyl-aryl, heteroaryl, or C₁₋₆ alkyl-heteroaryl,        wherein the cycloalkyl, heterocycloalkyl, heteroaryl, and aryl        groups are optionally substituted with a halogen, —NO₂, —OH,        —CN, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, or C₁₋₆        haloalkoxy. R² can be H, C₁₋₆ alkyl, or L-A. Radical L is a        linker and radical A is an active agent.

The compounds of formula I can include basic amino acids, such as thosehaving a positively charged side chain. Non-limiting examples of basicamino acids are Lys, Arg, HoArg, Agp, Agb, Dab, Dap and Orn, andstereoisomers thereof. The compounds of formula I can also includeacidic amino acids, such as those with a negatively charged side chain.Non-limiting examples of acidic amino acids are Asp, Glu, Aad, and Bec,and stereoisomers thereof. Basic amino acids can generally be referredby the symbol “X⁺” and acidic amino acids by “X⁻”. One of skill in theart will appreciate that other basic and acidic amino acids are known inthe art.

Amino acids useful in the compounds of the present disclosure includenaturally-occurring amino acids, as well as those amino acids that arelater modified, e.g., γ-carboxyglutamate and O-phosphoserine, as well asunnatural amino acids. Naturally-occurring α-amino acids include (shownwith the corresponding 3 letter and single letter codes), withoutlimitation, alanine (Ala, A), cysteine (Cys, C), aspartic acid (Asp, D),glutamic acid (Glu, E), phenylalanine (Phe, F), glycine (Gly, G),histidine (His, H), isoleucine (Ile, I), arginine (Arg, R), lysine (Lys,K), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), proline(Pro, P), glutamine (Gln, Q), serine (Ser, S), threonine (Thr, T),valine (Val, V), tryptophan (Trp, W) and tyrosine (Tyr, Y).Stereoisomers of a naturally-occurring α-amino acids include, withoutlimitation, D-alanine (DAla, a), D-cysteine (DCys, c), D-aspartic acid(DAsp, d), D-glutamic acid (DGlu, e), D-phenylalanine (DPhe, f),D-histidine (DHis, h), D-isoleucine (DIle, i), D-arginine (DArg, r),D-lysine (DLys, k), D-leucine (DLeu, 1), D-methionine (DMet, m),D-asparagine (DAsn, n), D-proline (DPro, p), D-glutamine (DG1n, q),D-serine (DSer, s), D-threonine (D-Thr, t), D-valine (D-Val, v),D-tryptophan (DTrp, w) and D-tyrosine (DTyr, y).

Unnatural amino acids include, without limitation, amino acid analogs,amino acid mimetics, synthetic amino acids, N-substituted glycines, andN-methyl amino acids in either the L- or D-configuration that functionin a manner similar to the naturally-occurring amino acids. For example,“amino acid analogs” are unnatural amino acids that have the same basicchemical structure as naturally-occurring amino acids, i.e., an α carbonthat is bound to a hydrogen, a carboxyl group, an amino group, but havemodified R (i.e., side-chain) groups. Suitable unnatural amino acidsinclude, without limitation, α-aminohexanedioic acid (Aad),α-aminobutyric acid (Abu), 3-aminobenzoic acid (3Abz),azetidine-2-carboxylic acid (Aca), 1-aminocyclobutane-1-carboxylic acid(Acb), α-amino-3-chloro-4,5-dihydro-5-isoazoleacetic acid (Acdi),4-amino-4-carboxy-1,1-dioxo-tetrahydrothiopyran (Acdt),1-amino-1-cyclohexane carboxylic acid (Ach),1-aminocyclopentane-1-carboxylic acid (Acp),1-aminocyclopropane-1-carboxylic acid (Acpc),4-amino-4-carboxytetrahydropyran (Actp),8-amino-1,4-dioxaspiro[4.5]decane-8-carboxylic acid (Aecc),(S)-2-amino-4-guanidino-butanoic acid (Agb), allylglycine (Agl),(S)-2-amino-3-guanidino-propanoic acid (Agp), 2-aminoheptanoic acid(Aha), 1-amino-1-(4-hydroxycyclohexyl) carboxylic acid (Ahch),α-aminoisobutyric acid (Aib), 2-aminoindane-2-carboxylic acid (Aic),1-amino-1-(4-ketocyclohexyl) carboxylic acid (Akch), 2-aminooctanoicacid (Aoa), 2-amino-2-naphthylacetic acid (Ana),1-amino-1-(3-piperidinyl)carboxylic acid (3Apc),1-amino-1-(4-piperidinyl)carboxylic acid (4Apc),2-amino-3-(4-piperidinyl) propionic acid (4App), homoarginine (HoArg),Nα-methyl-arginine ((NMe)Arg), Nα-methyl-aspartic acid ((NMe)Asp),α-aminooctanedioic acid (Asu),(R)-2-amino-3-(2-carboxyethylsulfanyl)propanoic acid (Bec),4,4′-biphenylalanine (Bipa),(R)-2-amino-3-(carboxymethylsulfanyl)propanoic acid (Bmc),4-carboxymethoxyphenylalanine (Bmp), 4-benzoylphenylalanine (Bpa),3-benzothienylalanine (Bta), 5H-thiazolo [3,2-a]pyridine-3-carboxylicacid (Btd), β-t-butyl-alaine (Bua), α-tert-butylglycine (Bug),4-cyano-2-aminobutyric acid (Cab), cyclobutylalanine (Cba),cyclohexylalanine (Cha), homocyclohexylalanine (HoCha),α-cyclohexylglycine (Chg), citrulline (Cit), homocitrulline (HoCit),cyclopropylalanine (Cpa), cyclopentylglycine (Cpeg), 3-carboxymethyl-1-phenyl-1,3, 8-tri azaspiro[4,5]decan-4-one (Cptd), homocysteine(HoCys), α,γ-diaminobutyric acid (Dbu), diethylglycine (Deg),3,3-diphenyl-alanine (Dpa), di-n-propylglycine (Dpg),α,β-diaminopropionic acid (Dap), α, γ-diaminobutyric acid (Dab),2-furyl-Alanine (Fua), homoarginine (HoArg), hydroxyproline (Hyp),O-benzyl-hydroxyproline (Hyp(Bzl)), homoleucine (HoLeu),2-Indanylglycine (Ing), methionine sulfoxide (Met(O)), methioninemethylsulfonium (Met (S-Me)), 3-(1-naphthyl)alanine (Nal1),3-(2-naphthyl)alanine (Nal2), 3-(carboxymethylamino)propanoic acid(Nglu), nipecotic acid (Nip), isonipecotic acid (IsoNip), norleucine(Nle), norvaline (Nva), octahydroindole-2-carboxylic acid (Oic),ornithine (Orn), 2-pyridylalanine (2Pal), 3-(3-pyridyl)alanine (3Pal),3-(4-pyridyl)alanine (4Pal), penicillamine (Pen), homophenylalanine(HoPhe), Nα-methyl-phenylalanine ((NMe)Phe), 2-chloro-phenylalanine(Phe(2Cl)), α-methyl-phenylalanine ((CαMe)Phe),3,4-dimethoxy-phenylalanine (Phe(3,4-di OMe)), 4-carboxyphenylalanine(Phe(4COOH)), 4-nitro-phenylalanine (Phe(4-NO₂)),4-trifluoromethyl-phenylalanine (Phe(4-CF₃)), 4-tert-butyl-phenylalanine(Phe(4-tBu)), 3,4-dichloro-phenylalanine (Phe(3,4-diCl)), phenylglycine(Phg), (2S,5R)-5-phenyl pyrrolidine-2-carboxylic acid (Ppca),propargylglycine (Pra), homoproline (HoPro), β-homoproline βHoPro),2-quinoylalanine (2Qa1), Nα-methylglycine (Sar), homoserine (HoSer),3-styryl-alanine (Sta), taurine (Tau), 4-thiazoylalanine (Tha),3-(2-thienyl)alanine (2Thi), 3-(3-thienyl)alanine (3Thi),thiazolidine-4-carboxylic acid (Thz), thiazolidine-2-carboxylic acid(Thz(2-COOH)), tetrahy dro-isoquinoline-3-carboxylic acid (3Tic),(R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic),3,5-dinitrotyrosine (Tyr(3,5di NO₂)), 3-nitrotyrosine (Tyr(3-NO₂)),3,5-diiodotyrosine (Tyr(diI)), and Na-methyl-valine ((NMe)-Val), aphenylalanine analog, derivatives of lysine, and stereoisomers thereof(see, Liu and Lam, Anal. Biochem., 295:9-16 (2001)). As such, theunnatural α-amino acids are present either as unnatural L-α-amino acids,unnatural D-α-amino acids, or combinations thereof.

The amino acids can also be categorized as basic, acidic, hydrophobicand/or polar. Some suitable basic amino acids of the disclosure are Lys,Arg, HoArg, Agp, Agb, Dab, Dap, Orn and stereoisomers thereof. Somesuitable acidic amino acids are Asp, Glu, Aad, Bec and stereoisomersthereof. Hydrophobic amino acids include, but are not limited to, Val,Leu, Ile, Met and Phe, and stereoisomers thereof. Polar amino acidsinclude, but are not limited to, Asn, Ser, Gln, Thr, and stereoisomersthereof.

Suitable phenylalanine analogs include, without limitation,homophenylalanine (HoPhe), phenylglycine (Phg), 3,3-diphenylalanine(Dpa), 4-aminophenylalanine (Phe(4-NH₂)), 2-methylphenylalanine(Phe(2-Me)), 3-methylphenylalanine (Phe(3-Me)), 4-methylphenylalanine(Phe(4-Me)), 4-azidophenylalanine (Phe(4-N₃)), 2-fluorophenylalanine(Phe(2-F)), 3-fluorophenylalanine (Phe(3-F)), 4-fluorophenylalanine(Phe(4-F)), 2-chlorophenylalmine (Phe(2-Cl)), 3-chlorophenylalanine(Phe(3-Cl)), 4-chlorophenylalanine (Phe(4-Cl)), 2-bromophenylalanine(Phe(2-Br)), 3-bromophenylalanine (Phe(3-Br)), 4-bromophenylalanine(Phe(4-Br)), 2-iodophenylalanine (Phe(2-I)), 3-iodophenylalanine(Phe(3-I)), 4-iodophenylalanine (Phe(4-I)),2-trifluoromethylphenylalanine (Phe(2-CF₃)),3-trifluoromethylphenylalanine (Phe(3-CF₃)),4-trifluoromethylphenylalanine (Phe(4-CF₃)), 2-methoxyphenylalanine(Phe(2-OMe)), 3-methoxy phenyl alanine (Phe(3-OMe)), 2-nitrophenyl almine (Phe(2-NO₂)), 3-nitrophenylalanine (Phe(3-NO₂)),4-nitrophenylalanine (Phe(4-NO₂)), 2-cyanophenylalanine (Phe(2-CN)),3-cyanophenylalanine (Phe(3-CN)), 4-cyanophenylalanine (Phe(4-CN)),3,4-dimethoxyphenylalanine (Phe(3,4-di OMe)), 3,4-difluorophenylalanine(Phe(3,4-di F)), 3,5-difluorophenylalanine (Phe(3,5-di F)),2,4-dichlorophenylalanine (Phe(2,4-diCl)), 3,4-dichlorophenylalanine(Phe(3,4-diCl)), 4-benzoylphenylalanine (Bpa), 4-carboxyphenylalanine(Phe(4COOH)), 4,4′-biphenylalanine (Bip),2,3,4,5,6-pentafluorophenylalanine (Phe(F₅)),3,4,5-trifluorophenylalanine (Phe(F₃)), 4-chlorophenylglycine(Phg(4-Cl)), 2-chlorophenylglycine (Phg(2-Cl)), 3-chlorophenylglycine(Phg(3-Cl)), 4-bromophenylglycine (Phg(4-Br)), 2-bromophenylglycine(Phg(2-Br)), 3-bromophenylglycine (Phg(3-Br)), 4-ethylphenylalanine(Phe(4-Et)), 4-ethoxyphenylalanine (Phe(4-OEt)), 4-butoxyphenylalanine(Phe(4-OBu)), O-methyltyrosine (Tyr(Me)), O-benzyltyrosine (Tyr(Bzl)),3,5-dibromotyrosine (Tyr(diBr)), 3,5-diiodotyrosine (Tyr(diI)),homotyrosine (HoTyr), 3-chlorotyrosine (Tyr(3-Cl)), stereoisomersthereof, and combinations thereof.

Suitable derivatives of lysine (Lys), ornithine (Orn) and Dbu, include,without limitation, Lys38, Lys27, Lys73, Lys55, Lys28, Lys72, Lys12,Lys123, Lys63, Lys124, Lys82, Lys31, Lys15, Lys125, Lys43, Lys24, Lys5,Lys4, Lys50, Lys81, Orn38, Orn27, Orn73, Orn55, Orn28, Orn72, Orn12,Orn123, Orn63, Orn124, Orn82, Orn31, Orn15, Orn125, Orn43, Orn24, Orn5,Orn4, Orn50, Orn81, Dbu38, Dbu27, Dbu73, Dbu55, Dbu28, Dbu72, Dbu12,Dbu123, Dbu63, Dbu124, Dbu82, Dbu31, Dbu15, Dbu125, Dbu43, Dbu24, Dbu5,Dbu4, Dbu50, Dbu81,stereoisomers thereof, and combinations thereof. See,Table 1 for a description of the structures for each of the lysinederivatives. Derivatives of Orn and Dbu are similar to the lysinederivatives with corresponding carboxylic acid attached to the sidechain of Orn and Dbu, respectively.

Suitable N-methyl amino acids include N-methyl-Ala, N-methyl-Cys,N-methyl-Asp, N-methyl-Glu, N-methyl-Phe, N-methyl-Gly, N-methyl-His,N-methyl-Ile, N-methyl-Arg, N-methyl-Lys, N-methyl-Leu, N-methyl-Met,N-methyl-Asn, N-methyl-Gln, N-methyl-Ser, N-methyl-Thr, N-methyl-Val,N-methyl-Trp, N-methyl-Tyr, N-methyl-Acp, N-methyl-Acb, N-methyl-Acpc,N-methyl-Cit, N-methyl-HoCit, N-methyl-Aad, N-methyl-4-Pal,N-methyl-3-Pal, N-methyl-Pra, N-methyl-Aib, N-methyl-Abu, N-methyl-Nva,N-methyl-Dpr, N-methyl-Dbu, N-methyl-Nle, N-methyl-Nal-2,N-methyl-Nal-1, N-methyl-Cha, N-methyl-Cpa, N-methyl-Hle,N-methyl-HoSer, N-methyl-Har, N-methyl-Hcy, N-methyl-Chg, N-methyl-Bta,N-methyl-2-Thi, N-methyl-3-Thi, N-methyl-Asu, N-methyl-Acdt,N-methyl-Ahch, N-methyl-Akch, N-methyl-Actp, N-methyl-Tyr(3-NO₂),N-methyl-Ach, N-methyl-3-Apc, N-methyl-4-Apc, N-methyl-4-App,N-methyl-Tha, N-methyl-Aoa, N-methyl-Aha, N-methyl-Orn, N-methyl-Aca,N-methyl-Agl, N-methyl-Cab, N-methyl-2-Pal, N-methyl-Cba,N-methyl-HoPhe, N-methyl-Phg, N-methyl-Phe(4-NH₂), N-methyl-4-Phe(4-Me),N-methyl-Phe(4-F), N-methyl-Phe(4-Cl), N-methyl-Phe(2-Br),N-methyl-Phe(3-Br), N-methyl-Phe(4-Br), N-methyl-Phe(3-CF₃),N-methyl-Phe(4-CF₃), N-methyl-Phe(4-NO₂), N-methyl-Phe(4-CN),N-methyl-Bpa, N-methyl-Phg(4-Cl), N-methyl-Phg(4-Br), N-methyl-Tyr(Me),N-methyl-Lys38, N-methyl-Lys27, N-methyl-Lys73, N-methyl-Lys55,N-methyl-Lys28, N-methyl-Lys72, N-methyl-Lys12, N-methyl-Lys123,N-methyl-Lys63, N-methyl-Lys124, N-methyl-Lys82, N-methyl-Lys31,N-methyl-Lys15, N-methyl-Lys125, N-methyl-Lys43, N-methyl-Lys24,N-methyl-Lys5, N-methyl-Lys4, N-methyl-Lys50, N-methyl-Lys81,N-methyl-Orn38, N-methyl-Orn27, N-methyl-Orn73, N-methyl-Orn55,N-methyl-Orn28, N-methyl-Orn72, N-methyl-Orn12, N-methyl-Orn123,N-methyl-Orn63, N-methyl-Orn124, N-methyl-Orn82, N-methyl-Orn31,N-methyl-Orn15, N-methyl-orn125, N-methyl-Orn43, N-methyl-Orn24,N-methyl-Orn5, N-methyl-Orn4, N-methyl-Orn50, N-methyl-Orn81,N-methyl-Dbu38, N-methyl-Dbu27, N-methyl-Dbu73, N-methyl-Dbu55,N-methyl-Dbu28, N-methyl-Dbu72, N-methyl-Dbu12, N-methyl-Dbu123,N-methyl-Dbu63, N-methyl-Dbu124, N-methyl-Dbu82, N-methyl-Dbu31,N-methyl-Dbul5, N-methyl-Dbu125, N-methyl-Dbu43, N-methyl-Dbu24,N-methyl-Dbu5, N-methyl-Dbu4, N-methyl-Dbu50, N-methyl-Dbu81,stereoisomers thereof, and combinations thereof.

Amino acid mimetics are chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunction in a manner similar to a naturally-occurring amino acid.Suitable amino acid mimetics include, without limitation, β-amino acidsand γ-amino acids. In β-amino acids, the amino group is bonded to theβ-carbon atom of the carboxyl group such that there are two carbon atomsbetween the amino and carboxyl groups. In γ-amino acids, the amino groupis bonded to the γ-carbon atom of the carboxyl group such that there arethree carbon atoms between the amino and carboxyl groups. Suitable Rgroups for β- or γ-amino acids include, but are not limited to,side-chains present in naturally-occurring amino acids and unnaturalamino acids.

N-substituted glycines are unnatural amino acids based on glycine, wherean amino acid side-chain is attached to the glycine nitrogen atom.Suitable amino acid side-chains (e.g., R groups) include, but are notlimited to, side chains present in naturally-occurring amino acids andside-chains present in unnatural amino acids such as amino acid analogs.Examples of N-substituted glycines suitable for use in the presentdisclosure include, without limitation, N-(2-aminoethyl)glycine,N-(3-aminopropyl)glycine, N-(2-methoxyethyl)glycine, N-benzylglycine,(S)—N-(1-phenylethyl)glycine, N-cyclohexylmethylglycine,N-(2-phenylethyl)glycine, N-(3-phenylpropyl)glycine,N-(6-aminogalactosyl)glycine, N-(2-(3′-indolylethyl)glycine,N-(2-(p-methoxy phenyl ethyl))glycine,N-(2-(p-chlorophenylethyl)glycine, andN-[2-(p-hydroxyphenylethyl)]glycine. N-substituted glycine oligomers,referred to herein as “peptoids,” have been shown to be proteaseresistant (Miller et al., Drug Dev. Res., 35:20-32 (1995)). As such,peptoids containing at least one unnatural α-amino acid, D-amino acid,or a combination thereof are within the scope of the present disclosure.

In still other embodiments, radicals R¹ and R² of formula I can eachindependently be H, C₁₋₆ alkyl or L-A. And radical L can be a linker andradical A can be an active agent.

In still other embodiments, R¹ can be acetyl, 3-amino propanoyl, Ebes,isobutyryl, valeryl, cyclohexyl acetyl, 5-bromo-2-furoyl, 3-phenylpropionyl, p-chlorophenyl acetyl, 4-nitrobezoyl, 3,5-dihydroxy beznoyl,4-(trifluoromethyl)benzoyl, 2-Methylthiazole-4-carbonyl, nicotinyl,2-naphthoyl, or biphenyl-4-carbonyl.

In some embodiments, radical X₂ of formula I can be Gly, Ala, Sar orβ-alanine, and stereoisomers thereof. Similarly, radical X₆ can be Val,Leu, Ile, Met, Phe, Asn, Glu, Gln, His, Lys, Arg, Asp, Gly, Ala, Ser,Thr, Tyr, Trp, Pro, Aad, Bec, Bmc, Bmp, Phe(4COOH), Hyp, HoSer, Tha,Ahch, Actp, Akch, Tyr(diI), Trp, Thz, 2Thi, 3Thi, Cit, HoCit, Aib, Nglu,or Fua, and stereoisomers thereof. In addition, radical X₇ can be Val,Leu, Ile, Met, Phe, Asn, Glu, Gln, His, Lys, Arg, Asp, Gly, Ala, Ser,Thr, Tyr, Trp, Pro, Bmp, HoSer, Nglu, HoCit, Bec, Aad, Hyp, Ahch,Phe(4COOH), Akch, Aecc, Abu, Phe(3,4-diOMe), Cpa, 2Thi, 3Thi, Thz, Phg,Phe(4-NO₂), Nle, (NMe)Phe, Aic, Chg, Bta, Bpa, Nal2, Nal1, Tic, Ppca,Cha, Bipa, Deg, Dpg, Acpc, Bmc, Cit, Sar, Tha, Pra, Actp, Aib, Agl,Acbc, Fua, Nva, Trp, Bug, Ach, (NMe)Val, Cpeg, (CαMe)Phe, Tyr(diI),Phe(2-Cl), Bua, HoPhe, HoLeu, Sta, Ing, Phe(4-CF₃), Oic, Dpa,Phe(4-t-Bu), HoCha or Phe(3,4-diCl), and stereoisomers thereof. In otherembodiments, each of radicals X₂, X₆ and X₇ can be a D-amino acid.

In other embodiments, the P2 peptide ligand of the scaffold of thepresent disclosure can have formula Ia (SEQ ID NO: 9):

Radicals R¹ and R² of formula Ia are as described above. In someembodiments, radical X₆ can be DSer, DAsp, Ahch, Bmp, DGlu, Nglu orDCit, and radical X₇ can be DPhe, DGlu, DSer, DBug, DBta, DVal,DAgIDPra, D(NMe)Val, D(CαMe)Val, DAbu, DIng, DIle, Actp, DTha, DAsp,DNal1 or Ppca. In some other embodiments, radical X₆ can be DAsp orDSer, and radical X₇ can be DGlu, DPhe, DSer, DVal, DBug or DBta.

R¹ and R² of formula Ia can each independently be H or C₁₋₆ alkyl. Insome embodiments, R¹ is H. In some embodiments, R² is H. In someembodiments, R¹ and R² are both H.

X₆ of formula Ia can be DSer, DAsp, DGlu, or DCit. X₇ of formula Ia canbe DPhe, DGlu, DSer, DBug, DBta, DVal, DAgl, DPra, D(NMe)Val,D(CαMe)Val, DAbu, DIng, DIle, DTha, DAsp, or DNal1. In some embodiments,X⁶ is DAsp and DSer. In some embodiments, X⁷ is DGlu, DPhe, DSer, DVal,DBug, or DBta.

In another embodiment, the P2 peptide ligand of the present disclosurecan be cGRGDsfc(SEQ ID NO: 13), cGRGDdfc(SEQ ID NO: 14), cGRGDsec(SEQ IDNO: 15), cGRGDdsc(SEQ ID NO: 16), cGRGDd-DBug-c(SEQ ID NO: 17),cGRGDd-DBta-c(SEQ ID NO: 18), cGRGDd-DBta-c(SEQ ID NO: 18), cGRGDdvc(SEQ ID NO: 12), CGRGDdvc(SEQ ID NO: 12), cGRGDdvC(SEQ ID NO: 12),CGRGDdvC(SEQ ID NO: 12), DPen-GRGDdv-DPen(SEQ ID NO: 19),DPen-GRGDdvc(SEQ ID NO: 20), cGRGDdv-DPen(SEQ ID NO: 21),Ac-cGRGDdvc(SEQ ID NO: 12), (β-alanine)-cGRGDdvc(SEQ ID NO: 22),(Ebes)-cGRGDdvc(SEQ ID NO: 12), caRGDdvc(SEQ ID NO: 23),c-Sar-RGDdvc(SEQ ID NO: 24), c-β-alanine-RGDdvc(SEQ ID NO: 25),cG-HoArg-GDdvc(SEQ ID NO: 26), cG-Agp-GDdvc(SEQ ID NO: 27),cG-Agp-GEdvc(SEQ ID NO: 28), cGRGDd-DAgl-c(SEQ ID NO: 29),cGRGDd-DPra-c(SEQ ID NO: 30), cGRGDd-DBug-c(SEQ ID NO: 17),cGRGDd-D(NMe)Val-c(SEQ ID NO: 31), cGRGDd-D(CαMe)Val-c(SEQ ID NO: 32),cGRGDd-DAbu-c(SEQ ID NO: 33), CGRGDd-DIng-c(SEQ ID NO: 34),c-Sar-RGD-Ahch-ic(SEQ ID NO: 35), c-Sar-RGD-Ahch-DBug-c(SEQ ID NO: 36),cGRGDd-DAgl-C(SEQ ID NO: 29), C-Sar-RGDd-DPra-C(SEQ ID NO: 37),C-Sar-RGDd-Actp-C(SEQ ID NO: 38), c-Sar-RGDd-DPra-C(SEQ ID NO: 37),c-Sar-RGDd-Actp-C(SEQ ID NO: 38), CGRGDd-DTha-C(SEQ ID NO: 39),cGRGDd-DPra-C(SEQ ID NO: 30), cGRGDd-Actp-C(SEQ ID NO: 40),c-Sar-RGD-Ahch-iC(SEQ ID NO: 35), c-Sar-RGD-Ahch-DBug-C(SEQ ID NO: 36),C-Sar-RGD-Bmp-dC(SEQ ID NO: 41), CGRGDe-Ppca-c(SEQ ID NO: 42),cGRGD-Nglu-Ppca-c(SEQ ID NO: 43), cGRGDd-DNal1-c(SEQ ID NO: 44) orcGRGDd-DBta-c(SEQ ID NO: 18). In other embodiments, the P2 peptide ofthe present disclosure can be cGRGDsfc(SEQ ID NO: 13), cGRGDdfc(SEQ IDNO: 14), cGRGDsec(SEQ ID NO: 15), cGRGDdsc(SEQ ID NO: 16), cGRGDdvc(SEQID NO: 12), cGRGDd-DBug-c(SEQ ID NO: 17) or cGRGDd-DBta-c(SEQ ID NO:18).

In another embodiment, the P2 peptide ligand of the present disclosurecan be cGRGDsfc(SEQ ID NO: 13), cGRGDdfc(SEQ ID NO: 14), cGRGDsec(SEQ IDNO: 15), cGRGDdsc(SEQ ID NO: 16), cGRGDdvc(SEQ ID NO: 12), CGRGDdvc(SEQID NO: 12), cGRGDdvC(SEQ ID NO: 12), CGRGDdvC(SEQ ID NO: 12),caRGDdvc(SEQ ID NO: 23), c-Sar-RGDdvc(SEQ ID NO: 24),c-β-alanine-RGDdvc(SEQ ID NO: 25), cGRGDd-DAgl-c(SEQ ID NO: 29),cGRGDd-DPra-c(SEQ ID NO: 30), cGRGDd-DBug-c(SEQ ID NO: 17),DPen-GRGDdv-DPen(SEQ ID NO: 19), DPen-GRGDdvc(SEQ ID NO: 20),cGRGDdv-DPen(SEQ ID NO: 21), cGRGDd-D(NMe)Val-c(SEQ ID NO: 31),cGRGDd-D(CαMe)Val-c(SEQ ID NO: 32), cGRGDd-DAbu-C(SEQ ID NO: 33),cGRGDdic, CGRGDd-Ding-c(SEQ ID NO: 34), c-Sar-RGD-Ahch-ic(SEQ ID NO:35), c-Sar-RGD-Ahch-DBug-c(SEQ ID NO: 36), cGRGDd-DAgl-C(SEQ ID NO: 29),C-Sar-RGDd-DPra-C(SEQ ID NO: 37), C-Sar-RGDd-Actp-C(SEQ ID NO: 38),c-Sar-RGDd-DPra-C(SEQ ID NO: 37), c-Sar-RGDd-Actp-C(SEQ ID NO: 38),CGRGDd-DTha-C(SEQ ID NO: 39), cGRGDd-DPra-C(SEQ ID NO: 30),cGRGDd-Actp-C(SEQ ID NO: 40), c-Sar-RGD-Ahch-iC(SEQ ID NO: 35),c-Sar-RGD-Ahch-DBug-C(SEQ ID NO: 36), C-Sar-RGD-Bmp-dC(SEQ ID NO: 41),CGRGDe-Ppca-c(SEQ ID NO: 42), CGRGD-Nglu-Ppca-c(SEQ ID NO: 43),CGRGDd-DNal1-C(SEQ ID NO: 44), CGRGD-D3Thi-Ppca-c(SEQ ID NO: 45),cGRGDd-DBta-c(SEQ ID NO: 18), cG-HoArg-GDdvc(SEQ ID NO: 26),cG-(NMe)Arg-GDdvc(SEQ ID NO: 46), cGR-Sar-Ddvc(SEQ ID NO: 47),cGRG-(NMe)Asp-dvc(SEQ ID NO: 48), cG-Agp-GDdvc(SEQ ID NO: 27),cG-Agp-GEdvc(SEQ ID NO: 28), cGRGDsdC(SEQ ID NO: 49), cGRGDd-Ding-c(SEQID NO: 34), cGRGDd-DNal1-c(SEQ ID NO: 44), cGRGDd-DNa12-c(SEQ ID NO:50), cGRGDd-D3Thi-c(SEQ ID NO: 51), cGRGDd-D2Thi-c(SEQ ID NO: 52),cGRGDdwc(SEQ ID NO: 53), cGRGDd-DTha-c(SEQ ID NO: 39),cGRGD-DCit-Ppca-c(SEQ ID NO: 54), cGRGDe-Ppca-c(SEQ ID NO: 42),cGRGD-NGlu-Ppca-c(SEQ ID NO: 43), cGRGD-DCit-DBta-c(SEQ ID NO: 55),cGRGD-DBec-Ahch-c(SEQ ID NO: 56), or cGRGD-DBec-DPra-c(SEQ ID NO: 57).In some embodiments, the peptide ligand is cGRGDdvc(SEQ ID NO: 12)(LXW7). In some embodiments, the P2 peptide ligand is peg2V (describedin further detail below).

The P2 peptide ligand can function to increase the attachment ofendothelial cells and/or endothelial progenitor cells. The P2 peptideligand can have an affinity for a cell surface integrin. The integrincan regulate retention, mobilization, vascularization, orendothelialization of cells. In some embodiments, the P2 peptide ligandbinds to one or more of integrins α4β1, α5β1, α6β3, αvβ3 and αvβ5. Insome embodiments, the P2 peptide ligand binds to integrin αvβ3 on thecells.

In some embodiments, the scaffold compound further includes a glycan,wherein each P1 subunit and each P2 subunit is linked to the glycan. Theglycan of the scaffold compound can be, for example, a glycosaminoglycanor a polysaccharide. The glycosaminoglycan can be selected from thegroup consisting of alginate, agarose, dextran, chondroitin, dermatan,dermatan sulfate, heparin, heparan sulfate, keratin, and hyaluronan. Inone embodiment, the glycan is selected from the group consisting ofdermatan sulfate, dextran, and heparin. In some embodiments, the glycanis dermatan sulfate (DS). Dermatan sulfate is a naturalglycosaminoglycan found mostly in skin, but also in blood vessels, heartvalves, tendons, lungs and intestinal mucosa. In addition to its role asa major constituent of the skin and other organs, dermatan sulfate isbelieved to play a part in repairing wounds, regulating the coagulationof blood, and responding to infections, though its role in theseprocesses is not well understood.

The P1 and P2 synthetic peptides can be directly linked to theglycosaminoglycan, or linked to the glycosaminoglycan via a linker. Thelinker can include one or more bivalent fragments selected independentlyin each instance from the group consisting of alkylene, heteroalkylene,cycloalkylene, cycloheteroalkylene, arylene, and heteroarylene, each ofwhich is optionally substituted. As used herein heteroalkylenerepresents a group resulting from the replacement of one or more carbonatoms in a linear or branched alkylene group with an atom independentlyselected in each instance from the group consisting of oxygen, nitrogen,phosphorus and sulfur.

IV. Pharmaceutical Compositions and Methods

The compounds described herein can be administered to a subject (e.g., apatient in need of treatment for a disease such as an ulcer, or in needof coronary stenting). In various embodiments, the compounds can beadministered intravenously or into muscle, for example. Suitable routesfor parenteral administration include intravascular, intravenous,intraarterial, intramuscular, cutaneous, subcutaneous, percutaneous,intradermal, and intraepidermal delivery. Suitable means for parenteraladministration include needle (including microneedle) injectors,infusion techniques, and catheter-based delivery.

Pharmaceutical compositions of any of the compounds described herein canbe formulated for parenteral administration or catheter-based delivery.For example, such compositions can include: a) a pharmaceutically activeamount of one or more of the compounds; b) a pharmaceutically acceptablepH buffering agent to provide a pH in the range of about pH 4.5 to aboutpH 9; c) an ionic strength modifying agent in the concentration range ofabout 0 to about 300 millimolar; and d) a water soluble viscositymodifying agent in the concentration range of about 0.25% to about 10%total formula weight; or any individual component a), b), c), or d); orany combinations of a), b), c) and d).

In various embodiments described herein, the pH buffering agents for usein the compositions and methods herein described are those agents knownto the skilled artisan and include, for example, acetate, borate,carbonate, citrate, and phosphate buffers, as well as hydrochloric acid,sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate,ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid,acetic acid, disodium hydrogen phosphate, borax, boric acid, sodiumhydroxide, diethyl barbituric acid, and proteins, as well as variousbiological buffers, for example, TAPS, Bicine, Tris, Tricine, HEPES,TES, MOPS, PIPES, cacodylate, or MES.

In various embodiments described herein, the ionic strength modifyingagents include those agents known in the art, for example, glycerin,propylene glycol, mannitol, glucose, dextrose, sorbitol, sodiumchloride, potassium chloride, and other electrolytes.

Useful viscosity modulating agents include but are not limited to, ionicand non-ionic water soluble polymers; crosslinked acrylic acid polymerssuch as the “carbomer” family of polymers, e.g., carboxypolyalkylenesthat can be obtained commercially under the CARBOPOL® trademark;hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;cellulosic polymers and cellulosic polymer derivatives such ashydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; gums suchas tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acidand salts thereof, chitosans, gellans or any combination thereof.Typically, non-acidic viscosity enhancing agents, such as a neutral orbasic agent are employed in order to facilitate achieving the desired pHof the formulation.

In various embodiments described herein, parenteral formulations can besuitably formulated as a sterile non-aqueous solution or as a dried formto be used in conjunction with a suitable vehicle such as sterile,pyrogen-free water. The preparation of parenteral formulations understerile conditions, for example, by lyophilization, can readily beaccomplished using standard pharmaceutical techniques available to thoseskilled in the art.

In various embodiments described herein, the solubility of compoundsused in the preparation of a parenteral formulation can be increased bythe use of appropriate formulation techniques, such as the incorporationof solubility-enhancing compositions such as mannitol, ethanol,glycerin, polyethylene glycols, propylene glycol, poloxomers, and othersknown to those of skill in the art.

In various embodiments described herein, formulations for parenteraladministration can be formulated to be for immediate and/or modifiedrelease. Modified release formulations include delayed, sustained,pulsed, controlled, targeted and programmed release formulations. Thus,one or more compounds can be formulated as a solid, semi-solid, orthixotropic liquid for administration as an implanted depot providingmodified release of the active compound. Illustrative examples of suchformulations include drug-coated stents and copolymeric(dl-lactic,glycolic)acid (PGLA) microspheres. In another embodiment, one or morescaffold compounds, or compositions comprising one or more scaffoldcompounds, can be continuously administered, where appropriate.

In any of the embodiments described herein, the compounds can beadministered alone or in combination with suitable pharmaceuticalcarriers or diluents. Diluent or carrier ingredients used in thecompound formulation can be selected so that they do not diminish thedesired effects of the compounds. The compound formulation can be in anysuitable form. Examples of suitable dosage forms include aqueoussolutions of the compounds, for example, a solution in isotonic saline,5% glucose, or other well-known pharmaceutically acceptable liquidcarriers such as alcohols, glycols, esters, and amides.

Suitable dosages of the compounds can be determined by standard methods,for example by establishing dose-response curves in laboratory animalmodels or in clinical trials. Illustratively, suitable dosages ofcompounds (administered in a single bolus or over time) include from 1ng/kg to about 10 mg/kg, 100 ng/kg to about 1 mg/kg, from about 1 μg/kgto about 500 μg/kg, or from about 100 μg/kg to about 400 μg/kg. In eachof these embodiments, dose/kg refers to the dose per kilogram of patientmass or body weight. In other illustrative aspects, effective doses canrange from about 0.01 μg to about 1000 mg per dose, 1 μg to about 100 mgper dose, or from about 100 μg to about 50 mg per dose, or from about500 μg to about 10 mg per dose, or from about 1 mg to 10 mg per dose, orfrom about 1 to about 100 mg per dose, or from about 1 mg to 5000 mg perdose, or from about 1 mg to 3000 mg per dose, or from about 100 mg to3000 mg per dose, or from about 1000 mg to 3000 mg per dose.

It is also contemplated that any of the formulations described hereincan be used to administer the compounds (e.g., one or more types) eitherin the absence or the presence of a catheter-based device. The compoundscan be formulated in an effective amount of an excipient. In any of theembodiments described herein, the excipient can have a concentrationranging from about 0.4 mg/mL to about 6 mg/mL. In various embodiments,the concentration of the excipient can range from about 0.5 mg/mL toabout 10 mg/mL, about 0.1 mg/mL to about 6 mg/mL, about 0.5 mg/mL toabout 3 mg/mL, about 1 mg/mL to about 3 mg/mL, about 0.01 mg/mL to about10 mg/mL, or about 2 mg/mL to about 4 mg/mL.

The dosage of the compounds can vary significantly depending on thepatient condition, the disease state being treated, the route ofadministration and tissue distribution, and the possibility of co-usageof other therapeutic treatments. The effective amount to be administeredto a patient is based on body surface area, patient weight or mass, andphysician assessment of patient condition. In various exemplaryembodiments, an effective dose can range from about 1 ng/kg to about 10mg/kg, from about 100 ng/kg to about 1 mg/kg, from about 1 μg/kg toabout 500 μg/kg, or from about 100 μg/kg to about 400 μg/kg. In each ofthese embodiments, dose/kg refers to the dose per kilogram of patientmass or body weight. In other illustrative aspects, effective doses canrange from about 0.01 μg to about 1000 mg per dose, from about 1 μg toabout 100 mg per dose, or from about 100 μg to about 50 mg per dose, orfrom about 500 μg to about 10 mg per dose, or from about 1 mg to 10 mgper dose, or from about 1 to about 100 mg per dose, or from about 1 mgto 5000 mg per dose, or from about 1 mg to 3000 mg per dose, or fromabout 100 mg to 3000 mg per dose, or from about 1000 mg to 3000 mg perdose. In any of the various embodiments described herein, effectivedoses can range from about 0.01 μg to about 1000 mg per dose, about 1 μgto about 100 mg per dose, about 100 μg to about 1.0 mg, about 50 μg toabout 600 μg, about 50 μg to about 700 μg, about 100 μg to about 200 μg,about 100 μg to about 600 μg, about 100 μg to about 500 μg, about 200 μgto about 600 μg, from about 100 μg to about 50 mg per dose, from about500 μg to about 10 mg per dose, or from about 1 mg to 10 mg per dose. Inother illustrative embodiments, effective doses can be 1 μg, 10 μg, 25μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 200 μg, 250 μg, 275 μg, 300μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 575 μg, 600 μg, 625 μg, 650μg, 675 μg, 700 μg, 800 μg, 900 μg, 1.0 mg, 1.5 mg, 2.0 mg, 10 mg, 100mg, or 100 mg to 30 grams.

Any effective regimen for administering the compounds can be used. Forexample, the compounds can be administered as a single dose, or as amultiple-dose daily regimen. Further, a staggered regimen, for example,one to five days per week can be used as an alternative to dailytreatment.

In any of the embodiments herein described, it is to be understood thata combination of two or more compounds, for example, differing in thepresence or absence of a glycan, can be used in place of a singlecompound.

Compounds can be sterilized before, during, and/or after formulation. Asused herein, “sterilization” or “sterilize” or “sterilized” refers todisinfecting the compounds by removing unwanted contaminants including,but not limited to, endotoxins and infectious agents.

In various illustrative embodiments, the compounds can be disinfectedand/or sterilized using conventional sterilization techniques includingpropylene oxide or ethylene oxide treatment, gas plasma sterilization,gamma radiation, electron beam, and/or sterilization with a peracid,such as peracetic acid. Sterilization techniques which do not adverselyaffect the structure and biotropic properties of the compounds can beused. Illustrative sterilization techniques include exposing thecompounds to peracetic acid, 1-4 Mrads gamma irradiation (or 1-2.5 Mradsof gamma irradiation), ethylene oxide treatment, sterile filtration, orgas plasma sterilization. In some embodiments, the compounds aresubjected to one or more sterilization processes. For example, thecompounds can be subjected to sterile filtration. The compounds can bedispensed into any type of container, which can be wrapped in a plasticwrap or a foil wrap, and can be further sterilized after such placementin a container.

The compounds can be combined with minerals; amino acids; sugars;peptides; proteins; vitamins (such as ascorbic acid); laminin; collagen;fibronectin; hyaluronic acid; fibrin; elastin; aggrecan; growth factors(such as epidermal growth factor, platelet-derived growth factor,transforming growth factor beta, or fibroblast growth factor);glucocorticoids such as dexamethasone; viscoelastic altering agents suchas ionic and non-ionic water soluble polymers; acrylic acid polymers;hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;cellulosic polymers and cellulosic polymer derivatives such ashydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose;poly(lactic acid); poly(glycolic acid); copolymers of lactic andglycolic acids; or other polymeric agents both natural and synthetic.

V. Kits

The compounds and compositions disclosed herein can be provided in oneor more types of kits. The kit can include packaging with one or morecontainers, at least one of which contains a scaffold compound. The kitcan also contain instructions for use of the components of the kit. Inone embodiment, the kit comprises one or more vessels, vials, orcontainers that hold one or more compounds. The kit can also include anyof the following components: one or more formulations or concentrations(dosages) of compounds, a buffer, a sterilizing or disinfecting agent, asyringe, a needle, proteins or polysaccharides, and/or instructionalmaterials describing methods for using the kit reagents. In any of theseembodiments, the kit can contain a component selected from the groupconsisting of a catheter, a stent, a balloon, and a combination thereof.The compounds can be lyophilized, for example, in a buffer or in water.

Articles of manufacture are also contemplated for any of theseembodiments. In any of the kit or article of manufacture embodimentsdescribed herein, the kit or article of manufacture can comprise a doseor multiple doses of the compounds. The compounds can be in a primarycontainer, for example, a glass vial, such as an amber glass vial with arubber stopper and/or an aluminum tear-off seal. In another embodiment,the primary container can be plastic or aluminum, and the primarycontainer can be sealed. In another embodiment, the primary container iscontained within a secondary container to further protect thecomposition from light.

In any of the embodiments described herein, the kit or article ofmanufacture can contain instructions for use. Other suitable kit orarticle of manufacture components include excipients, disintegrants,binders, salts, local anesthetics (e.g., lidocaine), diluents,preservatives, chelating agents, buffers, tonicity agents, antisepticagents, wetting agents, emulsifiers, dispersants, stabilizers, and thelike. These components can be available separately or admixed with thecompounds. Any of the composition embodiments described herein can beused to formulate the kit or article of manufacture.

VI. Embodiments

The following embodiments are contemplated. All combinations of featuresand embodiments are contemplated.

Embodiment 1: A compound comprising: one or more P1 subunits, wherein P1is a synthetic peptide comprising an amino acid sequence that comprisesa collagen-binding domain; one or more P2 subunits, wherein P2 is asynthetic peptide comprising an amino acid sequence that comprises anintegrin-binding domain; and a glycan, wherein each P1 subunit and eachP2 subunit is linked to the glycan.

Embodiment 2: An embodiment of embodiment 1, wherein P1 is a syntheticpeptide comprising an amino acid sequence having at least 80% sequenceidentity with the amino acid sequence RRANAALKAGELYKSILY(SEQ ID NO: 1).

Embodiment 3: An embodiment of embodiment 1, wherein P1 is a syntheticpeptide of up to 40 amino acids comprising the amino acid sequence

(SEQ ID NO: 1) RRANAALKAGELYKSILY.

Embodiment 4: An embodiment of any one of embodiments 1-3, wherein P2 isa synthetic peptide comprising an amino acid sequence that comprises anαvβ3-binding domain.

Embodiment 5: An embodiment of any one of embodiments 1-4, wherein P2 isLXW7.

Embodiment 6: An embodiment of any one of embodiments 1-4, wherein P2 ispeg2V.

Embodiment 7: An embodiment of any one of embodiments 1-6, wherein theglycan is a glycosaminoglycan or polysaccharide.

Embodiment 8: An embodiment of embodiment 7, wherein the glycan isselected from the group consisting of alginate, agarose, dextran,chondroitin, dermatan, dermatan sulfate, heparan, heparin, keratin, andhyaluronan.

Embodiment 9: An embodiment of embodiment 8, wherein the glycan isselected from the group consisting of dermatan sulfate, dextran, andheparin.

Embodiment 10: A compound comprising: one or more P1 subunits, whereinP1 is a synthetic peptide comprising an amino acid sequence thatcomprises a collagen-binding domain; and one or more P2 subunits,wherein P2 is a synthetic peptide comprising an amino acid sequence thatcomprises an integrin-binding domain.

Embodiment 11: An embodiment of embodiment 10, wherein P1 is a syntheticpeptide comprising an amino acid sequence having at least 80% sequenceidentity with the amino acid sequence RRANAALKAGELYKSILY(SEQ ID NO: 1).

Embodiment 12: An embodiment of embodiment 10, wherein P1 is a syntheticpeptide of up to 40 amino acids comprising the amino acid sequence

(SEQ ID NO: 1) RRANAALKAGELYKSILY.

Embodiment 13: An embodiment of any one of embodiments 10-12, wherein P2is a synthetic peptide comprising an amino acid sequence that comprisesan αvβ3-binding domain.

Embodiment 14: An embodiment of any one of embodiments 10-13, wherein P2is LXW7.

Embodiment 15: A composition comprising the compound of an embodiment ofany one of embodiments 1-14 and one or more pharmaceutically acceptableexcipients, diluents, or a combination thereof.

Embodiment 16: A method for improving endothelialization andvascularization of endothelial cells and/or endothelial progenitor cellsin a subject, the method comprising administering to the subject acomposition comprising the compound of an embodiment of any one ofembodiments 1-14.

Embodiment 17: An embodiment of embodiment 16, wherein the compositioncomprises one or more pharmaceutically acceptable excipients, diluents,or a combination thereof.

Embodiment 18: A method for improving endothelialization andvascularization of endothelial cells and/or endothelial progenitor cellsin a subject, the method comprising: administering to the subject afirst composition comprising the compound of an embodiment of any one ofembodiments 1-9; and administering to the subject a second compositioncomprising the compound of an embodiment of any one of embodiments10-14.

Embodiment 19: An embodiment of embodiment 18, wherein at least one ofthe first composition and the second composition comprises one or morepharmaceutically acceptable excipients, diluents, or a combinationthereof.

Embodiment 20: An embodiment of embodiment 18 or 19, wherein the secondcomposition is the first composition.

Embodiment 21: An embodiment of any one of embodiments 16-20, whereinthe subject is a patient suffering from a disease associated withendothelial dysfunction.

Embodiment 22: An embodiment of embodiment 21, wherein the disease is anulcer.

Embodiment 23: An embodiment of any one of embodiments 16-20, whereinthe subject is in need of coronary stenting.

VII. Examples

The present disclosure will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the disclosure in anymanner. Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1. Synthesis of SILY-DBCO

The synthetic scheme of SILY-DBCO is shown in FIGS. 1A and 1B.Fmoc-SILY-k was first synthesized by standard solid phase peptidesynthesis (SSPS) approach, followed by DBCO coupling in solution phasewith DBCO-OSu. Rink amide MBHA resin (1.0 g, 0.503 mmol, loading 0.503mmol/g) was swollen in DMF for 3 hours before Fmoc-deprotection.Protected Fmoc/Dde-SILY-k(Boc) beads were prepared with automatedpeptide synthesizer (CS-Bio). Five-fold excess of Fmoc-amino acids wereused for the coupling in presence of HCTU (6 eq.)/DIEA (12 eq.). Thecoupling times for the first 10 and the latter couplings were 2 hoursand 3 hours, respectively. The Fmoc was removed with 20%4-methylpiperidine twice (5 minutes, 15 minutes). The followingFmoc-amino acids were coupled sequentially: Fmoc-D-Lys(Boc)-OH,Fmoc-AEEA (SEQ ID NO: 58) linker, Fmoc-AEEA (SEQ ID NO: 58) linker,Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH,Fmoc-Lys(Dde)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Lys(Dde)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(pbf)-OH,Fmoc-Arg(Pbf)-OH. The beads were transferred to a 20-ml column from thesynthesizer. The N-terminal Fmoc and Dde groups were removed with 3%NH₂NH₂ in DMF (10 minutes, 15 minutes). After washing with DMF, MeOH,and DCM respectively, the free amino groups were re-protected with Fmocusing Fmoc-OSu (10 eq.) in presence of DIEA (20 eq.) in DCM. This stepwas repeated until a Kaiser test was negative. The resultingFmoc-protected SILY-k(Boc) beads were thoroughly washed with DMF, MeOHand DCM and then dried under vacuum for 1 hour before adding a cleavagecocktail of 82.5% TFA: 5% phenol: 5% thioanisole: 5% water: 2.5% TIS.The beads were rotated at room temperature for 4 hours. The liquid wascollected and concentrated by blowing with nitrogen gas. The crudeproduct was precipitated with cold ether and purified by reverse-phaseHPLC and lyophilized to give Fmoc-SILY-k in powder form. The identity ofthe compound was confirmed by MALDI-TOF MS, calculated forC₁₅₅H₂₁₈N₃₂O₃₇(m/z): 3119.61; Found: 3120.82 (MH⁺).

Fmoc-SILY-k (200 mg, 0.0577 mmol) was dissolved in anhydrous DMF (1 mL),then DBCO-OSu (25.6 mg, 0.06347 mmol) and DIEA (55 μL, 0.31735 mmol)were added to the solution. The reaction solution was stirred at roomtemperature for 1 hour. HPLC results indicated the completion ofcoupling. Cold ether was added to the liquid, and precipitate wascollected and redissolved in 25% 4-methylpiperidine (1 mL). The solutionwas stirred at room temperature for 1 hour. Cold ether was added toprecipitate the crude product which was purified by HPLC. The eluent waslyophilized to yield off-white powder SILY-DBCO. MALDI-TOF-MS,calculated for C₁₂₉H₂₀₁N₃₃O₃₃: 2740.51; Found: 2741.67 (MH⁺).

Example 2. Synthesis of LXW7-2N3

The synthesis of LXW7-2N₃ was achieved employing SPPS by CS Biosynthesizer as shown in FIG. 2 . The following Fmoc-amino acids werecoupled to Fmoc-deprotected Rink amide resin sequentially with HCTU/DIEAcoupling as described above: Fmoc-Lys(N₃)—OH, Fmoc-AEEA (SEQ ID NO: 58)linker, Fmoc-Lys(N₃)—OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-beta Ala-OH, Fmoc-beta Ala-OH,Fmoc-D-Cys(Trt)-OH, Fmoc-D-Val-OH, Fmoc-D-Asp(tBu)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Cys(Trt)-OH. Thelinear peptide was cleaved off the beads with TFA cocktail as mentionedabove. The linear crude peptide was precipitated with cold ether andcyclized with CLEAR OX resin in 50% 0.1 M ammonium acetate buffer inacetonitrile (ACN) for 3 hours (Ellman test was negative). The liquidwas collected and lyophilized to give the crude peptide which waspurified with HPLC. The eluent was lyophilized to give designed cyclicLXW7-2N₃. MALDI-TPF MS confirmed the structure. Calculated forC₉₁H₁₅₃N₃₃O₃₄S₂: 2316.07; Found: 2317.40 (MH⁺).

Example 3. Synthesis of LXW7-(SILY)₂ Conjugate

The LXW7-(SILY)₂ conjugate was synthesized by mixing the SILY-DBCO withLXW7-2N₃ in a mole ratio of 2:1 in acetonitrile/water (1:1) (FIG. 3 ).The resulting solution was stirred at room temperature for 3 h, then at4° C. overnight. The solution was directly submitted for HPLCpurification. The eluent was lyophilized to give LXW7-(SILY)₂ asoff-white powder.

Example 4. Synthesis of LXW7-DBCO

The synthetic route of LXW7-DBCO is shown in FIG. 4 . The synthesis ofLXW7-DBCO was achieved using a strategy similar to that of SILY-DBCO asdescribed above. The following Fmoc-amino acids were sequentiallycoupled to Fmoc-deprotected Rink amide resin: Fmoc-D-Lys(Boc)-OH,Fmoc-AEEA (SEQ ID NO: 58), Fmoc-D-Cys(Trt)-OH, Fmoc-D-Val-OH,Fmoc-D-Asp(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Gly-OH and Fmoc-D-Cys(Trt)-OH. Linear Fmoc-LXW7-k was cleaved offthe beads and purified by HPLC, followed by disulfide formation usingCLEAR OX beads in 50% 0.1 M ammonium acetate buffer in ACN for 3 hours(until Ellman test was negative). The liquid was collected andlyophilized to give crude Fmoc-LXW7-k which was further purified withHPLC. The eluent was collected and lyophilized to yield cyclicFmoc-LXW7-k as off-white powder. MALDI-TOF MS confirmed the identity.Calculated for C₅₆H₈₁N₁₅O₁₈S₂: 1315.53. Found: 1316.70 (MH⁺).

Fmoc-LXW7-k was then coupled with DBCO-OSu, followed byFmoc-deprotection and purification to give LXW7-DBCO, as described insynthesis of SILY-DBCO. MALDI-TOF MS calculated for C₆₀H₈₄N₁₆O₁₈S₂:1380.56. Found: 1381.80 (MH⁺).

Example 5. Synthesis of SILY-2N3

The synthesis of SILY-2N3 was similar to that of LXW7-2N3, except thatthe di-peptide linker of (beta Ala)-(beta Ala) in LXW7-2N3 was replacedwith GG in SILY-2N3. The scheme is shown in FIG. 5 . MALDI-TOF MScalculated for C₁₄₇H₂₄₈N₄₈O₄₃: 3373.87. Found: 3374.95 (MH⁺).

Example 6. Synthesis of SILY-(LXW7)₂ Conjugate

The SILY-(LXW7)₂ conjugate was synthesized by mixing the LXW7-DBCO withSILY-2N₃ in a mole ratio of 2:1 in acetonitrile/water (1:1) (FIG. 6 ).The resulting solution was stirred at room temperature for 3 hours, andthen at 4° C. overnight. The solution was directly submitted for HPLCpurification. The eluent was lyophilized to give SILY-(LXW7)₂ asoff-white powder.

Example 7. EC Attachment on Collagen Surface Modified with LXW7-SILY(Collagen-LXW7-SILY

Target culture wells in a 24-well plate were coated with 100 μg/mL RatTail Type I Collagen for 1 hour at 37° C. Collagen coated wells wererinsed 3 times with DPBS. LXW7-(SILY)2 or (LXW7)2-SILY was incubated inthe wells at a concentration of 50 μM for 1 hour at 37° C. The wellswere washed 3 times with DPBS and blocked with 1% BSA for 1 hour. Afterthe wells were rinsed 3 times with DPBS, 5×10⁴ human endothelialcolony-forming cells (HECFCs) suspended in the EGM-2 EC growth mediumwere added to the wells which were then incubated for 5 minutes at 37°C. and 5% CO₂. The wells were rinsed 3 times with DPBS to wash off theunattached cells, and the attached cells were fixed in 10% formalin for20 minutes. The cell nuclei were stained with DAPI and imaged using anOlympus IX81 microscope. Quantification of nuclei was performed usingthe ImageJ software (NIH).

As shown in FIG. 7 , HECFC attachment on collagen surface modified withLXW7-(SILY)2 or (LXW7)2-SILY was significantly higher than collagensurface without modification. HECFC attachment on (LXW7)2-SILY wassignificantly higher than on LXW7-(SILY)2, which indicated that(LXW7)2-SILY modification of collagen surface was more effective inimproving HECFC attachment.

Example 8. Effect of LXW7-SILY on EC Apoptosis Induced by Hypoxia

Target culture wells in a 96-well plate were coated with 100 μg/mL RatTail Type I Collagen for 1 hour at 37° C. Collagen coated wells wererinsed 3 times with DPBS. LXW7-(SILY)2 or (LXW7)2-SILY was added andincubated in the well at a concentration of 50 μM for 1 hour at 37° C.The wells were washed 3 times with DPBS and 2×10⁴ HECFCs suspended inthe EGM2 medium were added to the wells, and incubated overnight at 37°C. and 5% CO₂. The cells were transferred to 1% 02 and cultured for 48hours in EBM2 (EC basal medium without growth factors) or EGM2 (completeEC growth medium with all growth factors) respectively. Caspase 3activity assay was then performed to determine early apoptosis of thecultured cells according to the product manual.

As shown in FIG. 8 , compared with collagen surface withoutmodification, HECFCs cultured in EGM2 medium (in presence of VEGF) onLXW7-(SILY)2 or (LXW7)2-SILY modified collagen surface showed lowerCaspase 3 activity. (LXW7)2-SILY modification showed significant lowerCaspase 3 activity compared with the control collagen surface withoutmodification.

Example 9. EC Attachment on SIS Scaffold with LXW7-SILY

SIS scaffolds were cut by 12 mm biopsy punch and incubated with DPBS or50 μM (LXW7)2-SILY for 3 hours at 37° C. respectively. The scaffoldswere then rinsed 3 times with DPBS. Onto the scaffolds were added 2×10⁵HECFCs transduced with a GFP reporter suspended in 15 μL EGM-2 growthmedium, and the scaffolds were incubated for 10 minutes or 30 minutes at37° C. and 5% CO2, respectively. The scaffolds were washed 3 times withDPBS to wash off all the unattached cells, and the SIS/cell constructswere imaged using an Olympus IX81 microscope. As shown in FIG. 9 , atboth 10 minutes and 30 minutes after cell seeding, SIS scaffoldsmodified with (LXW7)₂-SILY (B, D) showed more attached HECFCs on thesurface than on the unmodified SIS scaffolds (A, C).

Example 10. EC Proliferation and Organization on SIS Scaffolds Modifiedwith LXW7-SILY

SIS scaffolds were cut by 12 mm biopsy punch and incubated with DPBS or50 NM (LXW7)2-SILY for 3 hours at 37° C. respectively. The scaffoldswere then rinsed 3 times with DPBS. Onto the scaffolds were added 2×10⁵HECFCs transduced with a GFP reporter suspended in 15 μL maintenancemedium, and the scaffolds were incubated for 1 hour at 37° C. and 5% CO2and added with 1 ml of EGM in each culture well. The scaffolds withcells were cultured at 37° C. and 5% CO2 for 7 days or 21 daysrespectively, and imaged using an Olympus IX81 microscope. As shown inFIG. 10 , compared with SIS scaffolds with modification (A, C), SISmodified with (LXW7)2-SILY supported more rigorous endothelial cellpopulation and denser vasculature (B, D).

Example 11. Synthesis of Pro-Angiogenic and Collagen-BindingPeptide-Hydrazides

Modified pro-angiogenic peptide-hydrazides of QK and LXW7 andcollagen-binding peptide-hydrazide SILY having sequences shown in Table1 were synthesized. The sequences of Table 1 are in 1-letter amino acidformat from N to C terminus, where upper case letters indicate L-aminoacids and lower case letters indicate unnatural D-amino acids, Acindicates acetylation of the N terminus, and aeea (SEQ ID NO: 58) is ashort (2-(2-aminoethoxy)ethoxy)acetic acid spacer (ChemPep, Inc.)resembling PEG₂.

TABLE 1 Peptide sequences Molecular Peptide Chemical Weight NameSequence Formula (g/mol) QK Ac-KLT WQE LYQ 1952.2 LKY KGI-amide(SEQ ID NO: 3) VEGFp KLT WQE LYQ LKY C₉₉H₁₅₆N₂₆O₂₆ 2126.5KGI GSG-hydrazide (SEQ ID NO: 4) Peg2V KLT WQE LYQ LKY C₁₁₁H₁₇₈N₂₈O₃₂2416.8 KGI-(aeea)₂-GSG- hydrazide (SEQ ID NO: 5) LXW7 CG RGD dvcC₅₄H₈₄N₁₈O₂₀S₂ 1369.5 -(aeea)₂-WG- hydrazide (SEQ ID NO: 10) SILYRRA NAA LKA GEL 2252.6 YKS ILY GSG- hydrazide (SEQ ID NO: 6)

A 2-chlorotrityl chloride resin (1.51 mmol/g, Anaspec) was rinsedsequentially 3 times with DMF, DCM, and DMF, and then swollen in 50%DCM/DMF for 1 hour. The resin was then reacted with 10% hydrazinehydrate (85%, Sigma) in synthesis grade DMF (Fisher) and 0.057 M DIPEA(Fisher) for 2 hours at room temperature while bubbling under nitrogen.The solution was drained and the resin was reacted again with freshsolution for 1 more hour, after which the resin was washed 3 times withDMF and reacted with 10% methanol in DMF to cap any remaining unreactedchloride groups. The hydrazide-resin was washed again 3 times with DMFand then reacted directly with the first amino acid (4 equivalents) withHOBt/HBTU (4 equivalents) and DIPEA (10 equivalents) bubbling undernitrogen overnight at room temperature. After coupling, resin was washedthoroughly with 2×DMF, 2×DCM, IPA, and then 2×DMF. Subsequent aminoacids were coupled for 20 minutes to 1 hour each at 50° C. on a LibertyBlue automated microwave peptide synthesizer (CEM) using 5 equivalentseach of Fmoc-amino acids (Aapptec), DIC, and OxymaPure with 0.1M DIPEAand deprotected with 20% piperidine in synthesis grade DMF for 3 minutesat 60° C. For N-terminal biotinylation, 5 equivalents of D-biotin(Anaspec) was coupled with 4 equivalents HBTU/HOBt and 10 equivalentsDIPEA overnight at room temperature. Peptides were cleaved for 3 hourswith 88% TFA, 5% phenol, 5% H₂O, and 2% TIPS and precipitated with colddiethyl ether. Crude peptides were redissolved in 5% acetonitrile andpurified to >90% purity through a C18 prep column (Spirit) against anacetonitrile (HPLC grade) gradient on an AKTApure 25 FPLC (GEHealthcare) and confirmed by MALDI-TOF mass spectrometry (Bruker).Before purification, cyclization of LXW7 was performed by oxidizingcysteine residues to form intramolecular disulfide bridges with ClearOxresin (Peptides International) according to the manufacturer's protocol.

Example 12. Peptide-Hydrazide Conjugation to Dermatan Sulfate

Carboxylic acids on dermatan sulfate (Celsus Laboratories, average MW46275) were activated with premium grade EDC (Thermofisher) for 5minutes in 0.1 M MES buffer with 8 M urea titrated to pH 4.5.Peptide-hydrazides were pre-dissolved in reaction buffer before additionto the activated DS solution with a final DS concentration of 10 mg/mL.After 2 to 48 hours, the reaction was stopped by titrating to pH 8 with0.5 M NaOH for 30 minutes. The product was then purified bysize-exclusion through 2 desalting columns (10 -mL Bio-gel P6 desaltingcartridges, Bio-Rad) in series on an AKTA purifier FPLC (GE Healthcare)and then lyophilized. For constructs with two different peptides,peptides were reacted sequentially with more EDC added upon conjugationof the second peptide, after taking a small sample of the reaction toquantify addition of the first peptide. Biotinylated molecules fordetection of surface binding were similarly synthesized by sequentiallyconjugating 1 equivalent of biotinylated peptide followed by the desiredequivalents of non-biotinylated peptide, taking into account conjugationefficiency. For example, a biotinylated DS-SILY4 was synthesized byreacting 1 equivalent of biotinylated-SILY-hydrazide followed by 4equivalents of SILY-hydrazide; a biotinylated DS-peg2V2 was synthesizedby reacting 1.33 equivalents of biotinylated-peg2V-hydrazide followed by4 equivalents of peg2V-hydrazide. DS control molecules were alsosynthesized by reacting DS under the same experimental conditions withand without EDC, labeled “EDC-activated DS” and “processed DS, no EDC”respectively, for 24 hrs but without peptide. These products were alsopurified by size-exclusion to remove buffer salts and any unreacted EDC.

Example 13. Characterization of Conjugated Peptides

All peptides were detected by concentration-dependent 280 nm absorbanceof aromatic amino acids on a NanoDrop One UV-Vis spectrophotometer(ThermoFisher). For increased specificity on molecules with multiplepeptides, peptide quantification was confirmed by detection of intrinsictryptophan (excitation at 295 nm, emission at 350 nm) and tyrosine(excitation at 280 nm, emission at 305 nm) autofluorescence on aSpectraMax M5 plate reader (Molecular Devices) in UV transparent 96-wellplates (Corning). All samples were prepared at 2 mg/mL in ultrapurewater and analyzed by taking the average of 5 repeated readings.

Circular dichroism spectra of peptides at 21.8 μM and constructs withequivalent total peptide concentrations were collected at roomtemperature in 0.01 M potassium phosphate buffer (pH 7.1) from 185 nm to260 nm with 4 accumulations at 50 nm/minute scan speed and 4 seconds DITin 1-mm quartz cuvettes (Starna) on a Jasco J-1500 CD Spectrophotometer.CD data was converted and presented as molar ellipticity indeg×cm²/dmol, [θ]=100*θ/(C×1), where θ is the degrees ellipticity, C isthe molar concentration, and 1 is the pathlength in cm. CD spectra ofconjugated peptides were calculated by collecting the spectra of theDS-peptide constructs and then subtracting out the signal for anequivalent concentration of EDC-reacted DS.

Two-dimensional nuclear magnetic resonance data were obtained in theform of high resolution 1H, 13C, and HSQC NMR spectra of 45 mg/mLdermatan sulfate in D₂O acquired for 6 hours on a Bruker instrument.Nanodrop absorbance spectra were obtained with 2 mg/mL samples evaluatedby absorbance spectral sweeps from 220 nm to 350 nm acquired on aNanoDrop One Spectrophotometer (ThermoScientific).

Example 14. LXW7-DS-SILY and LXW7(SILY)2 Effect on Platelet BindingUnder Flow

Flow kits from Ibidi (Martinsried, Germany) were used to measureendothelial recruitment under flow conditions. Microchannels were coatedwith equine fibrillar type I collagen (Chronolog, Havertown, PA) dilutedin 0.85% sodium chloride and incubated for one hour at room temperature.Unbound collagen was removed by rinsing three time with 1×PBS. Thetreatment molecule (DS-SILY, LXW7-DS-SILY, or LXW7-(SILY)2) wasincubated in the channel for 30 minutes at room temperature, and unboundpeptidoglycan was rinsed with 1× PBS.

Human microvascular endothelial cells purchased from Lonza were pushedthrough channels at a concentration of 6-8×10⁵ cells/mL by a syringepump at 2.3 μL/min. for a shear rate of 25 s⁻¹. After 30 minutes offlow, channels were rinsed with 100 μL, PBS to remove unbound cells.Adherent cells were stained with 10 μM CellTracker Green (ThermoFisherScientific) in media for 30 minutes at 37° C. Channels were rinsed with1× PBS and total fluorescence and cell number in the channels werequantified using ImageJ (NIH, Bethesda, MD).

Next, 15 mL of blood was drawn into sodium citrate vacutainers byvenipuncture and centrifuged for 20 minutes at 200 Gs and 37° C. and thetop layer of platelet-rich plasma (PRP) was collected. A syringe pumpwas used to flow PRP through microchannels at 4.2 mL/hr for a shear rateof 750 s⁻¹. After 4 minutes, the channels were rinsed with 100 μL, PBSto remove unbound cells. Adherent cells were stained with 10 μMCellTracker Green (ThermoFisher Scientific) in media for 30 minutes at37° C. Channels were rinsed with 1× PBS and total fluorescence and cellnumber in the channels were quantified using ImageJ (NIH, Bethesda, MD).

The results are presented in the graphs of FIG. 11 and. The data of FIG.11 demonstrates that LXW7-DS-SILY retains low platelet binding ofDS-SILY. The data of FIG. 12 demonstrates that LXW7-(SILY)2 (without DS)retains low platelet binding, but DS further reduces binding.

Example 15. Characterization of Molecule Binding to Collagen-CoatedSurfaces

High-binding 96-well plates (Corning) were coated with 50 μg/mL rat tailcollagen I (Corning) at 4° C. overnight or 2% reduced growth factorMatrigel (Corning #354230) in basal EBM media for 30 minutes at 37° C.and rinsed 3× with PBS before blocking non-specific binding with 1%Bovine Serum Albumin (BSA) for 1 hour at room temperature. Forfibrinogen surfaces, a fibrinogen coating solution of ˜2.75 mg/mLfibrinogen (Sigma) and 5.5 μg/mL aprotinin (Sigma) was incubated at 4°C. overnight in high-binding plates. Biotinylated samples dissolved in1% BSA were allowed to bind to the surface for 30 minutes at 37° C. on aplate shaker. After 3 washes with PBS, wells were incubated with 1:200streptavidin-HRP in 1% BSA, rinsed 3× with PBS, developed with a 1:1 Ato B color solution for 20 minutes, stopped with the addition 2N H₂SO₄,and read on a Spectramax M5 platereader for absorbance at 450 nm with540 nm correction.

Example 16. Cell Culture

Human Dermal Microvascular Endothelial Cells (HMVEC, Lonza CC-2543) andHuman Umbilical Vein Endothelial Cells (HUVEC, Lonza CC-2935) wereexpanded according to the manufacturers' protocol in EGM2-MV media andEGM-Plus media, respectively, and passaged at 70-90% confluence. Cellswere cryopreserved in 80% media, 10% sterile DMSO, and 10% FBS. Forexperiments, HMVECs were used between passages 6 and 8, and HUVECs wereused between passages 3 and 6 and maintained at 37C and 5% CO₂.

Example 17. Endothelial VEGFR2 Activation

HUVECs or HMVECs were seeded at 20000 cells per well in completeEGM-Plus growth media or complete EGM2-MV, respectively. Approximately24 hours after plating, the media was replaced with serum-free, growthfactor-free basal media (EBM) to serum-starve and synchronize the cells.The next day, the media was replaced with treatments dissolved in EBMfor 5 minutes and then immediately lysed with 50 μL, per well ofcomplete lysis buffer for 30 minutes on ice. Lysates were frozen at −80°C. until further analysis for phosphorylated VEGF receptor 2 and totalVEGF receptor 2 using a custom ELISA-type Multiplexed MesoScaleDiscovery (MSD) assay performed according to the manufacturer'sprotocol.

Example 18. Endothelial Proliferation in Response to Soluble Molecules

HMVECs were seeded at 2000 cells per well in a CELLBIND® 96-well plate(Corning) using complete EGM-2MV media. After 24 hours, media wasreplaced with EBM (serum and growth-factor free) for 4 hours and thenreplaced with treatments dissolved in EGM-2MV media lacking VEGF orlacking both FGF and VEGF. After 24 or 72 hours, proliferation wasevaluated by an endpoint MTS assay (CELLTITER 96® AQueous One Solution,Promega), by adding 10 μL, of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium(MTS) inner salt reagent to each well, incubating for 4 hours at 37° C.,and then measuring absorbance at 490 nm.

Example 19. Endothelial Proliferation in Response to Soluble Molecules

50 μL, treatments dissolved in 1% BSA in PBS were added to a BiocoatCollagen I 96-well plate (Corning) and incubated for 30 minutes at 37°C. before washing 3 times with PBS to remove unbound treatment. HMVECswere then seeded in either EGM2-MV media or EGM2-MV media without VEGFat a density of 1500 cells per well (low cell density) of 8000 cells perwell (high cell density). To evaluate initial attachment, media wasaspirated 5-8 hours after seeding and exchanged with alamarBlue® cellviability reagent (1:10, 110 μL/well, Molecular Probes) and incubatedfor 2.5 hours at 37° C. according to the manufacturers' protocol. 100 μLfrom each well was pipetted into a new plate and assayed forfluorescence at 560 nm excitation and 590 nm emission. Remaining reagentwas aspirated and replaced with fresh media and the alamarBlue®viability assay was performed every 2 days thereafter for 1 week. Tocontrol for initial differences in HMVEC attachment, proliferation datais presented as the alamarBlue® fluorescence signal normalized to thesignal at 8 hours.

Example 20. Endothelial Migration

ORIS 96-well plates (Platypus Technologies) were coated with 50 μg/mLhuman fibrillar collagen I (Chronolog) overnight at 4° C. Wells wererinsed 3× with PBS and left to air dry under UV for 1 hour beforeinserting ORIS silicone stoppers and seeding HMVECs at 8000 cells perwell around the stoppers. Cells were allowed to grow to confluence for48 hours before a 30 minute incubation with 4 μg/mL mitomycin C (Sigma)to halt proliferation. After removal of ORIS stoppers, cells and thecollagen-coated surface were incubated with 10 μM of DS-SILY constructsdissolved in EGM-2MV without VEGF for 15 minutes. The treatments wereremoved and replaced with growth media (EGM-2MV without VEGF) for 48hours before staining with 10 μM CellTracker Green (molecular probes)and fluorescent imaging on a Keyence BZ-X700 microscope. Fluorescentimages were quantified by pixel area analysis on ImageJ.

Example 21. Endothelial Tubulogenesis

Reduced growth factor Matrigel (Corning) was mixed with treatments onice to obtain the final desired concentrations (10, 20, and 30 μM)within the gel and distributed to Ibidi angiogenesis μ-slides at 10 μLper well to polymerize at 37° C. for 30 minutes. HMVEC cells were seededat 7500 cells per well in reduced growth factor media (EGM-2MV lackingFGF, VEGF, and IGF supplements). After 8 hours of incubation, cells werestained with 2 μM calcein AM and imaged on a Keyence BZ-X700 microscope.Image analysis tubule quantification was performed by AngioQuantsoftware with a prune factor of 25 to remove artefacts from cellulardebris.

Example 22. Ex Ovo Chick Chorioallantoic Membrane (CAM) Assay

Fertilized Hyline eggs obtained from the Avian Facility at UC Davis wereincubated at 37° C. for 72 hours with 6 rotations per day at 80%humidity in a Grumbach egg incubator. On day 3, eggs were cracked openinto disinfected weighboats (VWR) and covered with lids from squarepetri dishes (Fisher), and the embryos developed over a week in theincubator. On day 10, treatments encapsulated in collagen I gels weregently placed on the developing CAM. For experiments involvingtreatments freely eluting from the collagen gels, 100 μL of gel waspolymerized in a 10-mm diameter rubber mold against a sterilized glassslide, and 1 treatment gel and 1 control gel was placed per CAM. Thenumber of vessels in a 2-mm radius immediately surrounding the gel wasmanually counted in imageJ immediately after placement and 24 hourslater to determine changes surrounding vessels. The change in vesselsover 24 hours for treatment gels were normalized to the respectivechange in the control gel on the same developing embryo.

For experiments studying vascular invasion into collagen gels, 30 μLcollagen I gels with an embedded 3 mm×3 mm nylon mesh (Fisher) wereused. 4-5 gels were placed on each CAM, with 2-3 treatment gels of thesame type and 2 blank gels as internal controls to correct for embryovariability. In some embryos, the nylon meshes were dyed to improvevisualization of the mesh and vessels growing into the gel. Large (4cm×10 cm) nylon meshes were dyed by reacting in 226 mL of a 77 mM aceticacid (Sigma) solution with 10 drops of green food coloring (McCormick)or 250 μL of 3 mg/mL Evans blue (Alfa Aesar) at 121° C. for 15 minutesin an autoclave, and then cut to 3 mm×3 mm squares for embedding. Gelswere imaged daily through a Dino-Eye AM7025X camera installed on astereomicroscope; for images more than 2 days after gel implantation,0.25 mL PBS was pipetted on top of gels to help visualize vessels. Onday 16, 0.1 mL of 1 mg/mL RITC-dextran (70 kDa, Sigma) or Evans blue wasinjected into the CAM vasculature using a 30G needle, and the membranewas cut away from the embryo and placed in a petri dish for brightfieldand fluorescent imaging. To quantify vascularity within the gel between72-96 hours after gel placement, two independent observers scored eachgel for the proportion of grids within the mesh that showed positivevessel growth.

Example 23. Endothelial Monolayer Permeability

Using EGM2-MV media lacking VEGF, HMVECs were seeded onto 3-μm pore24-well transwell inserts (Corning) at 10,000 cells per insert. Thefollowing day, an additional 50,000 cells were seeded to each insert andallowed to grow for another 24 hours. Media was then removed andreplaced with 0.5 mL media to the bottom well and 0.1 mL of treatmentmedia to the insert for 10 minutes. Treatment was removed and 0.3 mL of1 mg/mL RITC-dextran in media was then added to each transwell insertand allowed to permeate for 30 minutes. Sample permeate was then assayedon a SpectraMax M5 plate reader for fluorescence (excitation: 570 nm;emission: 590 nm). RITC-dextran solutions and sample permeates were thenaspirated and replaced with another 0.1 mL of treatment media in thetranswell insert and 0.5 mL media in the bottom chamber. This procedurewas repeated daily over 4 days. Treatments were also compared to insertswith cells removed by rubbing away with a pipette tip to determine thebarrier efficacy of the monolayer and the degree of increasedpermeability.

Example 24. Design of Quantifiable Peptide Hydrazide Sequences withPreserved Post-Conjugation Bioactivity

Peptides were synthesized as C-terminal hydrazides to allow preferentialC-terminal EDC-conjugation over internal primary amines at low pH.Although unmodified QK and SILY peptides both contain multiple aromaticamino acids facilitating conjugated peptide quantification using 280 nmabsorbance and intrinsic autofluorescence, the original LXW7 peptidesequence needed to be further modified by addition of a C-terminaltryptophan for ease of downstream non-destructive quantification. Asexpected based on greater 280 nm absorbance for tryptophan compared totyrosine, we found that the 280 nm absorbance method was more sensitivefor quantification of QK-derived peptides and LXW7 than SILY.Non-destructive quantification of 2 different sequentially conjugatedpeptides by analyzing 280 nm absorbance of samples immediately beforeaddition of the second peptide provided adequate substitution estimatesunder the assumption that the conjugation reaction of the first peptidewas complete. We achieved more specific quantification of final peptidesubstitution on proteoglycan mimics containing both LXW7 and SILYmeasuring intrinsic autofluorescence of tryptophan (excitation 295 nm,emission 350 nm) and tyrosine (excitation 280 nm, emission 305 nm). Forthis particular peptide combination, the autofluorescence parametersallowed for specific detection of each peptide without significantsignal interference from the other peptide. Quantification of mimicswith both QK-derived peptides and SILY was also possible by theautofluorescence method, although the tyrosine signal includedcontributions from both peptides.

Example 25. Optimization of Peptide-Specific EDC-Hydrazide ConjugationChemistry

In the synthesis process, we discovered that peptide conjugationefficiency (defined as peptide equivalents successfully attached out oftotal reacted equivalents) to the dermatan sulfate backbone wasdependent on the peptide. Coupling of SILY was nearly 100% efficientafter 2 hours of reaction, while coupling of QK-derived peptides wasonly ˜70% efficient after a 4 hour reaction and coupling of LXW7suffered the lowest efficiency at ˜35-50% even after 48 hours ofreaction and additional EDC. In addition to peptide-specific conjugationefficiencies, we also found that comparable conjugation of SILYequivalents could be achieved with EDC activation of as few as 20% ofthe available carboxylic acids on DS (assuming approximately 100carboxylic acids per dermatan sulfate), whereas similar reduction of EDCsignificantly negatively impacted LXW7 conjugation. Given the vastdifferences in peptide conjugation efficiencies, it is likely thatstructural and steric effects affect the chemistry in asequence-specific manner.

We attempted to improve LXW7 coupling by using linear LXW7 in hopes ofreducing steric hindrance during conjugation and cyclizing the peptideafter conjugation. However, Ellman's assay revealed significantly fewerfree thiols than expected after conjugation and it was determined thatthe thiols in the linear peptide were consumed through irreversiblereaction with EDC. By adjusting several experimental parameters,including lengthening reaction time, better controlling pH, increasingpeptide equivalents, and adding more EDC over the course of thereaction, we achieved a maximum of 5.5 cyclized LXW7 peptides per DS. Indual peptide conjugation, we also discovered that the order of peptideaddition was crucial to achieving desired peptide substitution. SILYconjugation efficiency drastically decreased to 20-50% when it was addedas the second peptide (depending on the identity and quantity of thefirst peptide), while other peptides appeared to maintain relativelyconstant conjugation efficiency regardless of addition order; therefore,SILY peptides were added first in a dual peptide conjugation sequence.

Engineered molecules were given the following nomenclature to indicatethe type and number of conjugated peptides per DS:DS-(peptide)_(substitution #). For example, a decorin mimic composed ofa dermatan sulfate backbone and 4 SILY substitutions per DS is referredto as DS-SILY₄; a mimic with 3 peg2V peptides per DS is referred to asDS-(peg2V)₃. Pro-angiogenic decorin mimics are indicated as(peg2V)_(substitution #)-DS-SILY₄ or substitution #LXW7-DS-SILY₄ toemphasize the differing degrees of LXW7 substitution on LXW7-DS-SILY₄variants. Degrees of substitution are rounded to the nearest 0.5 forsimplicity.

Example 26. Characterization of Free and Conjugated Peptides

Circular dichroism spectra were measured to determine any peptideconformational changes upon spacer and hydrazide C-terminalmodifications and upon conjugation to dermatan sulfate. As seen in FIG.13A, circular dichroism spectra of modified peptides VEGFp and peg2Vvery closely matched the original alpha-helical QK signature withnegative peaks at 208 nm and 222 nm and a positive peak at 193 nm,despite hydrazide modification and the addition of GSG tripeptide andGSG-peg₂ C-terminal spacers for VEGFp and peg2V respectively.Importantly, as seen in FIG. 13E, VEGF-mimicking peptides alsomaintained the alpha-helical CD signature after conjugation to dermatansulfate, and the differences between the conjugated VEGFp and peg2V CDspectra were minimal; this is an essential finding since the closeproximity of the highly negatively charged dermatan sulfate moleculecould distort peptide secondary structure. In comparison to the alphahelical structure of VEGF-mimicking peptides, LXW7 had a slight negativesignal from 185 to 205 nm and almost no CD signal in other wavelengthsexamined, while SILY revealed a signature resembling a random coil of adenatured triple-helix with a minimum at 198 nm and low ellipticityabove 210 nm. Like free LXW7, conjugated LXW7 also had almost no CDsignal, although the minimal signal was slightly positive in the 185 to205 nm range (FIG. 13F). For SILY peptide, conjugation resulted in asignificantly more negative and slightly right shifted spectrum butstill lacking a positive signal in the 210 to 230 nm range, perhapsindicative of a slightly more organized collagen-like triple helix (FIG.13F).

In gathering the CD spectrum of the VEGFp conjugated to DS (denotedDSV), we discovered that lower substitution DSV₁ had a greater negativepeak around 200 nm than DSV₃ (FIG. 13C). This larger negative peak wasattributed to a higher concentration of DS, since the DSV₁ concentrationwas 3 times greater than DSV₃ in order to match samples for totalpeptide concentration. Upon background subtraction of the unmodified DSspectra, the resultant curves generally followed the expectedalpha-helical features but displayed unexpected positive features in the225 to 245 nm range. This led us to explore changes in the DS spectracoming from the EDC reaction. In this investigation, we observed anunexpected change in the CD spectra of EDC-reacted DS, indicating a GAGstructural/conformational change induced by the EDC reaction (FIG. 13B);the CD spectrum had pronounced deepening of the negative peak around 200nm and a new positive peak at 235 nm, most closely resembling the CDspectra of intact collagen triple helices although the positive peak isslightly more right-shifted. Since the products were purified bysize-exclusion prior to CD analysis, these changes could be attributedto covalent addition of EDC or co-purified EDC due to strongassociation.

13C and 1H NMR analysis revealed multiple new carbons and protons on DSafter the EDC reaction (FIGS. 14A and 14B). Additionally, absorbancespectral sweeps demonstrated increased absorbance at 220 nm proportionalto the amount of reacted EDC, likely due to new imide bonds (resemblingamide bonds). The CD, NMR, and absorbance data together indicate thatEDC reacted covalently and irreversibly through O-acylisourearearrangement to stable N-acylurea after carboxylic acid activation(FIG. 15 ). Irreversible N-acylurea formation with EDC has been welldocumented, and is likely more abundant in the given reaction due to thelack of NHS although the molar ratio of EDC to carboxylic acid groupswas approximately 1:1. Due to modification of the carboxylic acid, theN-acylurea EDC adduct would reduce the overall negative charge on DS atneutral pH and potentially thereby cause a conformational change andaffect its electrostatic interactions with other biological molecules.

Example 27. Relative Surface-Binding of Molecules to Various CoatedSurfaces

The ability of DS-SILY₄ to bind to various protein surface coatings wasinvestigated using streptavidin-HRP detection of bound biotinylatedDS-SILY₄ molecules. Here we studied collagen and fibrinogen bindingproperties of the proteoglycan mimics since this proposed therapy isdesigned to target angiogenic activity to the wound site by tethering toextracellular matrix. Using standard surface coating procedures forcollagen (50 μg/mL) and matrigel (2%), ˜7.5 μg/cm² rat tail collagen Iand ˜28 μg/cm² matrigel were allowed to bind to a high bind plate. Basedon Corning's reported composition of matrigel (60% laminin, 30% collagenIV, and 8% entactin), the amounts of collagen I versus collagen IV inthe standard coating protocols should be comparable and thus the resultsof this experiment were assumed to reflect differential binding ofmolecules to collagen I versus collagen IV and laminin.

FIG. 16A shows that DS-SILY₄ bound in a dose-dependent manner tocollagen I, collagen IV/laminin, and fibrinogen with binding capacitiesof 1.6, 1.1, and 0.30 AU respectively. FIG. 16A also shows that bindingdetection on a plate coated with collagen following our reportedprotocol was roughly equivalent to the binding on a manufacturercollagen coated plate (Corning Biocoat) up to 1 μM of DS-SILY₄. In theranges of 1 to 10 μM, DS-SILY₄ exhibited a negative binding trend on theBiocoat plate, likely resulting from preferential association ofDS-SILY₄ with itself over the surface at higher concentrations. Asimilar downward binding trend was also seen for DS-SILY₄ on thecollagen-coated plate in the 5 to 10 μM range. Differing collagensurface densities may account for these observed differences in bindingbehavior. Fibrinogen surfaces were exposed to ˜400 μg/cm² fibrinogen.Despite the high coating density, the fibrinogen surfaces exhibited alow level of dose dependent DS-SILY₄ binding relative to collagen I andmatrigel surfaces (FIG. 16A).

As seen in FIGS. 16A and 16B, peg2V conjugation to DS-SILY₄ increasedbinding to collagen I and matrigel. To determine whether this increasewas caused by non-specific binding due to greater peptide substitutionor intrinsic collagen I affinity, we studied whether removing the knowncollagen-binding SILY peptide would still allow for dose-dependentcollagen I binding. FIG. 16B reveals that, when conjugated to DS, peg2Vdoes have very high intrinsic collagen I affinity but appears toaggregate more readily at concentrations above 0.5 μM, represented by asignificant drop in binding at high concentrations. In fact, between theconcentrations of 0.05 to 1 μM, DS-(peg2V)₂ had the highest collagen Ibinding capacity of all the molecules studied. Interestingly, sinceDS-(peg2V)₂ exhibited higher collagen I binding capacity than DS-SILY4,collagen may have more binding sites for peg2V than SILY. This isespecially significant given that the addition of only 2 peg2V peptidesproduced greater binding than 4 SILY peptides. Notably, FIG. 16B alsodemonstrates that DS-(peg2V)₂ has a higher affinity for collagen I thanDS-SILY₄, while their matrigel binding affinities were similar (FIG.16C); however it appears that the combination of peg2V with SILYsynergistically strengthens binding affinity to both collagen I andmatrigel since (peg2V)₂-DS-SILY₄ demonstrated the highest collagen I andmatrigel affinities, with significantly greater binding at lowerconcentrations <0.05 μM. The data taken altogether suggests that peg2Vexhibits specific collagen I binding even greater than SILY, but thesimultaneous conjugation of both peptides increases collagen I and IVaffinity and reduces molecular aggregation high concentrations.

As seen in FIGS. 16B and 16C, LXW7 conjugation to DS-SILY₄ alsoincreased binding to collagen I and matrigel. Taking a differentapproach to investigating the effects of LXW7 on collagen binding, wecompared variants with differing degrees of LXW7 substitution.3LXW7-DS-SILY₄ had greater binding capacity than 1LXW7-DS-SILY₄ at allconcentrations and both bound collagen I better than DS-SILY₄ alone,suggesting that the addition of LXW7 increased collagen I binding sites.From FIG. 16B, addition of LXW7 also improved molecule affinity tocollagen I since 3LXW7-DS-SILY₄ had significantly greater affinity than1LXW7-DS-SILY₄. Most notably, as seen in FIGS. 16C and 16D, a higherLXW7 substitution also improved collagen I specificity over matrigelsince 3LXW7-DS-SILY₄ had higher collagen I binding but lower matrigelbinding than 1LXW7-DS-SILY₄. It is possible that LXW7 does this by morespecifically binding collagen I or that it imparts non-specific collagenI binding and simultaneously hinders matrigel binding by unknownmechanisms, but biotinylated DS-LXW7 variants (lacking SILY) were notsynthesized to rule out non-specific binding.

To study the deterioration of binding activity over time, we observedsurface binding properties of (peg2V)₂-DS-SILY₄ and DS-SILY₄ after 2weeks in 1% BSA at room temperature (FIG. 16F). Comparing FIGS. 16E and16F, it is clear that the collagen I and matrigel binding curves shiftedafter the 2 week incubation such that higher concentrations are neededto achieve the same binding for peg2V₂-DS-SILY₄. Although this shift wasalso observed for DS-SILY₄ to collagen I interaction, the degradedDS-SILY₄ binds matrigel even more than the freshly dissolved treatment.The observed concentration shifts suggest that the effective moleculeconcentration has decreased after the 2 week incubation, which could becaused by degradation of the full molecule or hydrolysis of thecollagen-binding SILY peptide. FIG. 16F also shows that DS-SILY4 hadincreased matrigel binding capacity after the 2 week incubation,indicating that partially degraded DS-SILY has increased matrigelbinding capacity, while its affinity remained constant.

Example 28. HMVEC Attachment to Surface-Bound Molecules

Microvascular endothelial cell adhesion to surface-bound molecules wasstudied for its relevance to endothelial recruitment in the woundhealing process, with results shown in FIG. 17 . At low cell densities,both 1 μM and 10 μM DS-SILY₄ surface treatments caused a slight 7%decrease in HMVEC attachment compared to a BSA blocked collagen surface,although the decrease was only statistically significant at 10 μM(asterisks indicate treatments that were statistically different fromDS-SILY₄ control). Since DS-SILY₄ binds and masks collagen fibrils,decreased adhesion is likely due to a decrease in exposed collagen andtherefore reduced cell adhesion sites. Surprisingly, both 1 μM and 10 μM(peg2V)₃-DS-SILY₄ caused an even greater 15% decrease in HMVECattachment compared to DS-SILY₄ alone. This additional decrease could beattributed to increased surface concentrations of peg2V3-DS-SILY₄ due tohigher surface binding discovered through the binding experiments (FIG.16 ), thereby masking the underlying collagen more effectively andfurther decreasing adhesion sites for cells. Alternatively, it ispossible that the peg2V peptide prevents cell attachment by unknownmechanisms.

To determine the effects of LXW7 conjugation on cell adhesion, multiplevariants with varying degrees of LXW7 substitution were investigated. Alow LXW7 substitution of 1 caused a similarly significant 18% decreasein cell adhesion at both 1 μM and 10 μM concentrations; however,increasing LXW7 substitution caused a trending increase in HMVECattachment, with the greatest positive effect at 3.5 LXW7. At 1 μM, itappears that a threshold substitution degree of at least 2 LXW7 isrequired to prevent negative effects on attachment, and all othersubstitutions tested (2, 4, and 5.5) have no significant effects onattachment at compared to DS-SILY₄. At 10 μM, a moderate degree ofsubstitution between 3 and 4 provided optimal attachment significantlygreater than DS-SILY₄ alone by 8% and comparable to attachment to anuntreated collagen I surface blocked with 1% BSA (FIG. 17A). Celladhesion was similar between the 1 and 10 μM concentrations for alltreatments (FIG. 17C). Given the LXW7 peptide's role facilitating celladhesion through integrin-binding, it was surprising that surfacecoatings with increased LXW7 substitution beyond 3.5 did notsignificantly increase cell attachment when cells were seeded sparsely.However, increasing the cell density more than 5 fold was able to revealthese expected increases in cell binding as seen in the 4LXW7-DS-SILY₄variant and restored HMVEC attachment comparable to an unblockedcollagen I surface.

Example 29. HMVEC Proliferation Responses to Soluble Molecules (MTSAssay) Vs. Surface-Bound Molecules (Alamar Assay)

Initial investigation of proliferative bioactivity using an endpoint MTSmetabolic assay (FIG. 18 ) revealed a trending increase in HMVECproliferation in response to constant 24 hour and 72 hour stimulation by10 μM soluble DS(peg2V)₃ and (peg2V)₃-DS-SILY₄ but not DSV₃ whencompared to DS alone. The trending increase in bioactivity forDS(peg2V)₃ compared to DSV₃ suggests that the longer spacer between theactive QK sequence and the DS backbone improved the proliferativeresponse, perhaps by allowing better access for peptide binding to theVEGF receptor or by better conserving the alpha-helical secondarystructure. Furthermore, since free (peg2V)₃-DS-SILY₄ in solution wouldalso bind any secreted collagen I and other extracellular matrixmolecules to an extent, as seen in the binding experiments (FIG. 16 ),(peg2V)₃-DS-SILY₄ may be present at higher local concentrations aroundthe cell when bound and could therefore exert a more potentproliferative response from (peg2V)₃-DS-SILY₄ in comparison toDS(peg2V)₃. Finally, 10 μM soluble DS(peg2V)₃ and (peg2V)₃-DS-SILY₄ alsosignificantly increased proliferation compared to 10 μM solubleDS-SILY₄, indicating that the conjugated peg2V peptide was responsiblefor the proliferative response. Since LXW7-DS-SILY₄ variants requiredsurface binding to enact proliferative bioactivity throughintegrin-binding, the LXW7-conjugated variants were not tested in thesoluble proliferation assay.

Both soluble and bound forms of molecules would be present in a dynamicequilibrium in this initial experimental design and the two forms mayproduce differing effects, and thus we sought to investigate the effectsof bound molecules alone. Furthermore, the bioactivity of acollagen-bound form would more directly reflect the application of atargeted pro-angiogenic therapy in a chronic wound. To investigate theeffects of surface-bound molecules, collagen surfaces were firstincubated with treatments to bind and then washed thoroughly to removeany unbound molecules before seeding and following cell growth everyother day for a week with media replenished every other day. To controlfor differences in initial cell number due to surface effects on cellattachment, signals were normalized to the initial signal 8 hours afterseeding.

From the results shown in FIG. 19 , DS-SILY₄ surface treatmentssignificantly improve endothelial proliferation compared to untreatedcollagen alone. The effects of 1 μM DS-SILY₄ were only statisticallysignificant over no treatment when compared in media lacking VEGF, but10 μM DS-SILY₄ significantly improved proliferation in media with andwithout VEGF. The effects of 1 and 10 μM concentrations were verycomparable, because although 10 μM DS-SILY₄ improved proliferation over1 μM by day 6 in media with VEGF, 1 μM DS-SILY₄ was equally as effectiveas 10 μM during the first 4 days of growth. The relative similaritybetween the 1 and 10 μM DS-SILY₄ concentrations could be expected giventhe apparently saturated collagen I-binding plateau found in the surfacebinding experiments (FIG. 16 ).

A single peg2V conjugated DS-SILY₄ variant was investigated in 2concentrations to observe proliferative effects. In media lacking VEGF,(peg2V)₃-DS-SILY₄ did not have any significant proliferative effects,although the higher 10 μM concentration showed a trending improvement inproliferation (FIG. 19A). In contrast, (peg2V)₃-DS-SILY₄ surfacetreatment significantly improved proliferation over DS-SILY with VEGFsupplementation, indicating an apparent synergistic effect of theconjugated peg2V with VEGF (FIG. 19B). At 1 μM, (peg2V)₃-DS-SILY₄surface treatment was as effective as the 10 μM treatment, bothresulting in ˜9-fold increase after 144 hours of growth; however, the 1μM more effectively potentiated VEGF activity compared to 10 μM,providing a 27% increase in proliferation over the 1 μM DS-SILY₄ controlversus an 18% increase over the 10 μM DS-SILY₄ control.

Multiple LXW7-DS-SILY₄ variants with differing degrees of substitutionwere tested to determine the effect of LXW7 density on endothelialproliferation, since the varying surface densities could affect integrinbinding conformation and subsequent VEGF receptor clustering andactivation. Results are shown in FIGS. 20 and 21 . In media lacking VEGFand low 1 μM concentration, HMVECs exhibited a peculiar bimodalproliferative response to increasing LXW7 substitution with peaks at2-3.5 LXW7 and 5.5 LXW7. However, at 10 μM, all substitutions less than5.5 resulted in significantly increased HMVEC proliferation with themost significant activity at 3.5 LXW7. Interestingly, these variantswere as effective as the 10 ng/mL VEGF supplementation, causing a 6-foldincrease in proliferation, and the 3.5 LXW7 variant appeared to be evenmore effective than 10 ng/mL VEGF (although the increase was notstatistically significant). With VEGF supplementation and at low 1 μMconcentration, proliferation was greatest at 1 and 5.5 LXW7, but onlystatistically different than DS-SILY₄ when the degree of substitutionexceeded 4. Similarly at 10 μM, the effects of degrees of substitutionbelow 5.5 were also statistically equivalent to DS-SILY₄, but the5.5LXW7-DS-SILY₄ showed significantly increased HMVEC proliferation overthe DS-SILY₄ control. Based on this data, LXW7 synergy with VEGF (10ng/mL based on manufacturers specifications) only appeared to be presentfor the 5.5LXW7-DS-SILY₄ variant, whereas this concentration of VEGFactually seemed to mask the effects of low LXW7 substitution. Takentogether, the results indicated that high LXW7 substitution variantswere more effective at potentiating VEGF at the 10 ng/mL testedconcentration, while lower LXW7 substitution optimal at 3.5 exertedsignificant proliferative bioactivity without exogenous VEGF. At 1 μM,effects were variable but tended to be most effective around 2 and 5.5LXW7 in a bimodal manner in media conditions both with and without VEGF.These results suggest that an optimal substitution of approximately 3LXW7 per DS may promote optimal proliferative bioactivity throughintegrin clustering.

Example 30. HMVEC Migration Responses to Surface-Bound Molecules (ORISAssay)

Migration responses were explored with and without the application ofthe anti-proliferative agent Mitomycin C (MMC) in the ORIS assay tostudy closure of a circular breach in a confluent endothelial monolayer.When cells were exposed to MMC in the absence of VEGF, Peg2V and LXW7conjugated to DS-SILY₄ both significantly stimulate endothelialmigration more than DS-SILY₄ alone (FIG. 22 ). Without MMC treatment,cells appearing in the previously obstructed central region could beattributed to both migration and proliferation over 48 hours; in thisscenario, there were no statistically significant differences in cellcoverage between the variants, although the 4LXW7-DS-SILY4 stimulatedslightly greater cell coverage. Nevertheless, the differences inmonolayer closure with and without MMC suggest that the gap closure wasmainly due to DS-SILY4 is primarily a proliferative response, whilecoverage from (peg2V)3-DS-SILY4 was from a migratory response and4LXW7-DS-SILY4 had important contributions from both migration andproliferation. The migration data without MMC closely matched theobserved 48 hour proliferative responses to surface-bound moleculesdiscussed previously in our study; in the absence of VEGF,(peg2V)3-DS-SILY4 stimulated proliferation about the same degree asDS-SILY4 over an untreated collagen surface, while 4LXW7-DS-SILY4 wasable to produce a greater proliferative response.

Example 31. HMVEC 2D Tubulogenesis in Response to Matrigel EmbeddedMolecules

In vitro matrigel tubulogenesis assays revealed that treatments embeddedwithin gels at a 10 μM concentration had no significant effects on HMVECtubule number, length, size, or number of junctions compared to theuntreated controls (FIG. 23 ). Increasing the embedded treatmentconcentration to 20 μM resulted in increased tubule number but trendingdecreases in length, size, and junction number (FIG. 24 ). Since thisassay evaluates the responses of cells seeded on top of the gels, thedata suggests that the 10 μM concentration may not present an adequatesurface concentration to elicit a detectable response. Moreover whilethe rapid time course of the matrigel assay with tubules forming withina few hours and then destabilizing after 12 hours allows for quickscreening of angiogenic factors, it may only reflect very acute cellularchanges and their corresponding immediate effects on tubule formation.Nevertheless, it was clear from the matrigel assay that the vascularnetwork formed differed in structure compared to untreated controls.

Example 32. Changes in Chick Chorioallantoic Membrane Vascularization inResponse to Collagen Gels: Eluted Vs. Embedded Tethered Molecules

FIG. 25 shows that 100 μM free VEGFp and QK peptides had comparablevasculogenic activity to each other and to 2 μg/mL VEGF-A, stimulatingacute increases (30-50% compared to matched control gels on the sameegg) in the number of blood vessels in a 2-mm radius surroundingimplanted 10-mm diameter gels after 24 hours (n≥5). In contrast, elutionof 100 μM LXW7 from collagen gels either decreased or caused no changesin surrounding vessel growth after 24 hours (FIG. 25 shows a decreasethat is not statistically significant). The lack of increasedvascularity in response to free LXW7 seen here suggests that the LXW7peptide needs to be surface-bound to promote angiogenesis.

Both 10 μM and 20 μM DS-SILY₄ showed a very slight increase in gelvascularity that was not statistically significant over the blankcontrols (FIGS. 26A and 26B). However, FIG. 26C demonstrates that 10 μMgel-embedded (peg2V)₃-DS-SILY₄ significantly increased the vascularizedarea within the gel compared to matched blank controls by day 4 afterimplantation. As can be seen in the representative vascularized gelimages (FIG. 27 ), new vessels invaded the collagen gels in a radialfashion with many vessels going towards and then away from the center ofthe transplanted gel. Under high magnification through a stereoscope,rapidly flowing red blood cells were apparent within the invadingcapillaries with directional flow indicative of the presence of both newarterioles and venules. Since 10 μM (peg2V)₃-DS-SILY₄ already showed asignificantly improved vascular response, the 20 μM concentration wasnot investigated.

Both 10 μM and 20 μM 4LXW7-DS-SILY₄ embedded gels caused a largeincrease in vascularized gel area, but not statistically significantthan the organism-matched blank controls (FIGS. 26D and 26E). In fact,10 μM and 20 μM 4LXW7-DS-SILY₄ treatments produced almost identical gelvascularization results. Interestingly, the proportion of vascularizedgel area in 4LXW7-DS-SILY₄ treated gels was similar to that seen for(peg2V)₃-DS-SILY₄ treated gels, but the organism-matched blank controlsexhibited an trending increase in vascularity compared to blank controlson eggs treated with DS-SILY₄ and (peg2V)₃-DS-SILY₄. Since the increasein the blank controls was not present in the DS-SILY₄ or(peg2V)₃-DS-SILY₄ treated eggs, it is more likely that this unexpectedincrease in the blank control gels results from an induced a systemicresponse perhaps by release of angiogenic factors into the bloodcirculation rather than 4LXW7-DS-SILY₄ releasing from the collagen gel.

In an attempt to better visualize new vasculature invading the implantedcollagen gels, we injected ˜100 μL 1 mg/mL high molecular weightfluorescent dextran (70 kDa RITC-dextran) into the CAM vessels 7 daysafter transplantation, cut out integrated collagen gels and surroundingmembrane, and imaged with fluorescence microscopy. For proof-of-concept,control CAMs without transplanted gels were injected and imaged withthis method and demonstrated that the fluorescent dextran reasonablyrepresented the vasculature without leaking outside the vessels (FIG.28A). In our studies, both treatment and blank collagen gels showeddiffuse red fluorescence in all eggs (FIG. 28B). This fluorescent signalwas not present in blank collagen gels not exposed to RITC-dextran (datanot shown). Since all the collagen gels exhibited some degree ofleakiness measured by the RITC-dextran fluorescence, it is difficult toinfer the permeability effects of the collagen-bound treatmentmolecules. Injection of 100 μL of a 2% w/v Evans blue in PBS for bettervisualization of extravasate suggested that while all the collagen gelshad some accumulation of the blue dye, the (peg2V)₃-DS-SILY₄ gels lookeddarker indicating increased vascular permeability in surroundingvessels.

Example 33. Effects of Soluble VEGF-Mimicking Molecules on HMVECMonolayer Permeability (Transwell)

Given that the CAM assay suggested that the tested molecules may bealtering vascular permeability and that VEGF is also known as vascularpermeability factor and is hence widely known to increase vascularpermeability, we were interested in the effects of VEGF-mimickingpeptides on endothelial permeability in comparison to VEGF. We thereforefollowed treatments after 10 minutes through 3 days, although it isworth noting that our experimental method effectively investigatedpermeability effects after a series of repeated bolus treatments ofsoluble molecules on a HMVEC monolayer. The transwell results in FIG.29A demonstrate that the seeded HMVECs indeed formed a monolayersignificantly decreasing permeation of the 70 kDa RITC-dextran comparedto wells with no cells, while FIGS. 29A and 29C indicate thatEDC-reacted DS had no effects on permeability. FIG. 29B shows that asshort as 10 minute incubation of 10 ng/mL VEGF, 30 μM QK, and 10 μMDS-(peg2V)₃ increased monolayer permeability compared to the non-treatedmedia (EGM2-MV lacking VEGF) and 10 μM EDC-reacted DS, although only theQK and DS-(peg2V)₃ groups showed statistically significant increases inour experiment. After 18 hours, the increases in VEGF- and QK-inducedpermeability had disappeared, while DS-(peg2V)₃ still maintainedsignificantly increased permeability (FIG. 29B). By 24 hours, thepermeability of DS-(peg2V)₃ treated wells recovered to control levels(FIG. 29B). By extending treatments out to 48 and 72 hours, both theVEGF and DS-(peg2V)₃ treatments actually showed trending decreases inmonolayer permeability (FIG. 29C). Both VEGF and DS-(peg2V)₃ weresignificantly different from untreated controls by 72 hours, and theeffect of 10 μM DS-(peg2V)₃ was statistically equivalent to 10 ng/mLVEGF (FIG. 29C).

Example 34. Additional Supporting Data

Data have demonstrated that arterial mimics can be developed in thelaboratory using human SMCs alone or with ECs. With the appropriateculture media components, the tissue cultured mimics can be induced tobehave as the healthy or diseased arterial state. An important findingfrom our study is that quiescent (or contractile) SMCs support adhesionof a monolayer of ECs, while the proliferative (or hyperplastic) SMCs donot support the same EC monolayer. This unique culture system providesthe opportunity to optimize the design of our therapies to preventplatelet binding to the damaged vessel wall and/or support EPC captureand monolayer formation on the medial mimetics as the mimetics can bemade to behave as denuded diseased vessels or as recently denudedhealthy vessel. The cultures can be maintained for over one month andcan serve as a high throughput method for testing various densities ofpeptide grafted to the DS and multiple therapeutic concentrations. Amodel is shown pictorially in FIG. 30 .

The antithrombotic proteoglycan mimetic, DS-SILY, binds to collagen withhigh affinity, such that it remains bound during blood flow, andprevents collagen-induced platelet activation in vitro. Platelet bindingand activation on a collagen surface was inhibited up to nearly 90% withDS-SILY treatment. Further, DS-SILY was found to promote EC migration upto 32% compared to control collagen surfaces, presumably through its DSbackbone, which can bind to and activate resident FGF-2 and FGF-10.Therefore, DS-SILY blocks platelet binding and supports EC growth,resulting in faster vessel healing.

An In vivo Ossabaw pig angioplasty model was used to assess plateletdeposition (acute) and neointimal hyperplasia (1-month recovery) withDS-SILY treatment. Porous angioplasty balloons were employed fordelivery of soluble DS-SILY to the target arteries in the peripheralvasculature of pigs. Renal, femoral, and iliac arteries were denuded byballoon expansion, followed by delivery through the porous balloon.Arteries were treated with and without the use of a bare metal stent andthe healing response was assessed for neointimal hyperplasia.

Arteries for assessing the acute response of balloon injury wereharvested within hours of angioplasty. The arteries were rinsed withsaline and fixed in 10% formalin overnight, and prepared for SEMvisualization. FIG. 31 shows that platelets are scarcely found on thedenuded artery in DS-SILY treatment, compared to significant plateletcoverage of the artery wall in sham (saline) control treated arteries.High magnification images in FIG. 31 show numerous projections andspreading of platelets on sham control arteries, indicative of plateletactivation. In contrast, when platelets were found present on theDS-SILY treated artery, they remained largely rounded. These resultsindicate that DS-SILY binds to the exposed collagen of the vessel walland prevents platelet binding and activation, the first steps that leadto thrombosis and intimal hyperplasia.

Neointimal hyperplasia was assessed by histology after 1-month recoveryby measuring the distance between the internal elastic lamina and thelumen, or between a stent post and the lumen (FIG. 32 ). Neointimalhyperplasia is observed with and without stents in sham controls,whereas minimal intimal hyperplasia is observed in DS-SILY treatedarteries. With respect to DS-SILY, these data show that it suppressesSMC migration and proliferation, two hallmarks of neointimalhyperplasia. Also, DS-SILY suppresses inflammatory cytokine production,a second hallmark in intimal hyperplasia. DS-SILY also suppresses, butdoes not eliminate protein production by SMCs. In vivo data (FIG. 32 )shows that DS-SILY suppresses platelet binding to denuded blood vesselsand inhibits intimal hyperplasia at 4 weeks as compared to untreatedvessels.

Using primary artery-derived ECs and blood-derived EPCs as living probesto screen One-Bead-One-Compound (OBOC) combinatorial peptide libraries,several high-affinity binding ligands against the α_(v)β₃ integrin onthe surface of EPCs/ECs were identified. One of these peptides, LXW7, acyclic octapeptide (cyclic cGRGDdvc (SEQ ID NO: 11)), was found to bindstrongly to primary EPCs/ECs, but weakly to platelets and does not bindto THP-1 monocytes. The LXW7 peptide, when grafted to surfaces, supportsadhesion and spreading of EPCs and human coronary ECs, but not plateletsor monocytes. Culture plates, 24-well, were coated with 500 μL of 20μg/mL Avidin for 1 hour at 37° C. followed by washing with PBS andcoating with either 500 μL molar equivalents of D-biotin, LXW7-biotin,or GRGD (SEQ ID NO: 2)-biotin. After washing with PBS and blocking with1% BSA wells were ready for cell attachment with 5×10⁴ HCAECs or THP-1monocytes suspended in the respective maintenance medium, or freshlyisolated platelets at a density of 5×10⁷ platelets/cm² incubated for 16h at 37° C., 5% CO₂. The cells were washed with PBS, and fixed in 10%formalin for 20 min. For evaluation of HCAEC, the cells were blockedwith 1% BSA and incubated overnight with mouse anti-CD31 antibody(1:100; Abcam) in 1% BSA at 4° C., followed by probing with goatanti-mouse Alexa Fluor 594 conjugate in 1% BSA. Nuclei were stained withDAPI. Images were captured using an Olympus IX81 microscope. Phasecontrast images were taken for evaluation of THP-1 monocyte and plateletattachment. Quantification of images was performed using the Image Jsoftware (NIH). Representative images are shown in FIG. 33A, andquantification is shown in FIG. 33B. LXW7 peptide supported robust HCAECbinding, but not monocyte or platelet binding, while the RGD peptidegenerally supported monocyte and platelet binding.

To further probe the effects EC and EPC binding to surface-grafted LXW7,in vitro studies were performed to investigate ERK1/2 and VEGF receptorphosphorylation as both are downstream markers of EC binding through theαvβ3. LXW7 stimulated cell proliferation (FIG. 34A) and phosphorylationof ERK ½ and VEGF2 receptor (FIG. 34B), as expected due to adhesion viathe αvβ3 integrin and further suggests the specificity of LXW7. To testthe ability of LXW7 to promote endothelialization, we evaluatedpolymer-based small diameter vascular grafts (ID 1 mm) functionalizedwith LXW7 via Click chemistry in a rat carotid artery bypass model (FIG.35 ). We found that LXW7-modified grafts showed significantly higherpatency rate than the control grafts. After 6 weeks post implantation, 5out of 6 LXW7-modified grafts were patent versus only 1 out of 6 controlgrafts was patent (FIG. 35 ). In addition, mature ECs were presentthroughout the whole length of the LXW7-modified grafts while only alimited number of ECs were identified in the middle segment of thecontrol grafts. This confirms that LXW7 coating on the luminal surfaceof synthetic vascular grafts can generate a “living” endothelium.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purpose of clarity of understanding,one of skill in the art will appreciate that certain changes andmodifications may be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

1-22. (canceled)
 23. A method for improving endothelialization andvascularization of endothelial cells and/or endothelial progenitor cellsin a subject, the method comprising administering to the subject acomposition comprising a first compound, the first compound comprising:one or more P1 subunits, wherein P1 is a synthetic peptide comprisingthe amino acid sequence RRANAALKAGELYKSILY (SEQ ID NO: 1) or aconservatively modified variant sequence having at least 80% sequenceidentity with the amino acid sequence RRANAALKAGELYKSILY (SEQ ID NO: 1);and one or more P2 subunits, wherein P2 is cGRGDdvc (LXW7), cGRGDsfc,cGRGDdfc, cGRGDsec, cGRGDdsc, cGRGDd-DBug-c, cGRGDd-DBta-c, Ac-cGRGDdvc,(β-alanine)-cGRGDdvc, (Ebes)-cGRGDdvc, cGRGDd-DAgl-c, cGRGDd-DPra-c,cGRGDd-D(NMe)Val-c, cGRGDd-D(CαMe)Val-c, cGRGDd-DAbu-c, cGRGDd-DNal1-c,cGRGDd-DNa12-c, or peg2V.
 24. The method of claim 23, wherein P1 is asynthetic peptide comprising the amino acid sequence RRANAALKAGELYKSILY(SEQ ID NO: 1).
 25. The method of claim 23, wherein P1 is a syntheticpeptide of up to 40 amino acids comprising the amino acid sequenceRRANAALKAGELYKSILY (SEQ ID NO: 1).
 26. The method of claim 23, whereinP2 is LXW7.
 27. The method of claim 23, wherein P2 is peg2V.
 28. Themethod of claim 23, wherein the method further comprises administeringto the subject a second compound comprising: one or more P1 subunits;one or more P2 subunits; and a glycan, wherein each P1 subunit and eachP2 subunit is linked to the glycan.
 29. The method of claim 28, whereinthe glycan is a glycosaminoglycan or polysaccharide.
 30. The method ofclaim 29, wherein the glycan is alginate, agarose, dextran, chondroitin,dermatan, dermatan sulfate, heparan, heparin, keratin, or hyaluronan.31. The method of claim 30, wherein the glycan is dermatan sulfate,dextran, or heparin.
 32. The method of claim 23, wherein the compositioncomprises one or more pharmaceutically acceptable excipients, diluents,or a combination thereof.
 33. The method of claim 23, wherein thesubject is a patient suffering from a disease associated withendothelial dysfunction.
 34. The method of claim 33, wherein the diseaseis an ulcer.
 35. The method of claim 23, wherein the subject is in needof coronary stenting.
 36. A surface modified with a compound comprising:one or more P1 subunits, wherein P1 is a synthetic peptide comprisingthe amino acid sequence RRANAALKAGELYKSILY (SEQ ID NO: 1) or aconservatively modified variant sequence having at least 80% sequenceidentity with the amino acid sequence RRANAALKAGELYKSILY (SEQ ID NO: 1);and one or more P2 subunits, wherein P2 is cGRGDdvc (LXW7), cGRGDsfc,cGRGDdfc, cGRGDsec, cGRGDdsc, cGRGDd-DBug-c, cGRGDd-DBta-c, Ac-cGRGDdvc,(β-alanine)-cGRGDdvc, (Ebes)-cGRGDdvc, cGRGDd-DAgl-c, cGRGDd-DPra-c,cGRGDd-D(NMe)Val-c, cGRGDd-D(CαMe)Val-c, cGRGDd-DAbu-c, cGRGDd-DNal1-c,cGRGDd-DNa12-c, or peg2V.
 37. The surface of claim 36, wherein P1 is asynthetic peptide comprising the amino acid sequence RRANAALKAGELYKSILY(SEQ ID NO: 1), and wherein P2 is LXW7 or peg2V.
 38. The surface ofclaim 36, wherein the compound further comprises a glycan, and whereineach P1 subunit and each P2 subunit is linked to the glycan.
 39. Thesurface of claim 36, wherein the surface comprises cells attached to thesurface, the cells comprising endothelial cells and/or endothelialprogenitor cells.
 40. The surface of claim 36, wherein the surfacecomprises collagen.
 41. The surface of claim 40, wherein the surfacecomprises porcine small intestinal submucosa (SIS).
 42. The surface ofclaim 36, wherein the surface is a luminal surface of a vascular graft.